**2. Materials and Methods**

The survey was designed according to a study conducted in a Chinese university [22], the Generalized Anxiety Disorder 7-Item Scale (GAD-7) [23] and the guiding principles of emergent psychological crisis intervention in COVID-19 [24]. Information such as age, gender, grade, health state, residence, annual household income and grade were collected using single item measures. Anxiety was obtained according to GAD-7 and Cronbach- s alpha coefficient was 0.901. Invulnerability, conformity, insensitivity, rebelliousness and bravado were measured by a Likert-type scale of 1–4; "1 = Never or very rare", "2 = Sometimes", "3 = Often" and "4 = Very often or always". The risk level of the living region was decided according to the data from the National Health Commission of the People's Republic of China (a high risk region ≥ 50 confirmed cases in two weeks; the moderate risk region had < 50 confirmed cases and had new confirmed cases in two weeks; the mild risk region had no new confirmed case in two weeks). The cross-sectional survey was carried out from 1 April to 1 June 2020. The Questionnaire Star (https://www.wjx.cn, accessed on 30 June 2020) was used as the platform of this survey. The participants were recruited from the Wuhan University of Science and Technology (WUST) and the website of the survey was shared in WeChat or a QQ group of the students of WUST. Before the survey, an electronic informed consent was signed online. The participants were informed that the survey was anonymous and they could reject the survey for any reason. The study was approved by the Ethics Committee of Wuhan University of Science and Technology

(protocol code 20200407, 4 February 2021). All data were analyzed by SPSS software and a one-way analysis of variance was applied to assess the significance of each factor.

#### **3. Results**


*2.2.2 Structural-visual method in linguistics*

For most people instead of a speech mechanism a translation mechanism works introduced by the school approach to language learning, which is physiologically a different process, the opposite of language. To start the speech mechanism, you need to create a database of sound images, speech-motor images and direct connections of these images with meanings.

The translation mechanism prevents the creation of such links and leads to the formation of links between the signs of one and another language. Therefore, after school experience even in non-translation courses on the communicative method or when using programs like "Rosetta stone" most students choose their usual translation strategy. At the same time, acts of thinking and understanding are performed in the native language, and externally language actions (listening, speaking, and communication) in the target language.

Teachers do not yet have the tools to identify, measure, and change the type of mental process, and to find out what process is actually going on. And it is almost impossible to explain these subtleties to the student in order to obtain self-esteem, especially since these processes are unconscious and do not lend themselves to conscious regulation. The relations between processes in reality, sensory and motor processes in the psyche and language are extremely difficult to explain and poorly understood, and have long been the subject of disputes and disagreements between various sciences and scientific directions. Therefore, consciously explaining to the student what, how and why he needs to do is not a solution to the problem. As a result, the habit of the student, developed earlier in the course of a long experience of studying at school or other institutions, still takes over.

Psycholinguistic studies have shown that the use of grammatical rules for planning and controlling utterances slows down speech activity [3], since the same areas of the brain and mental processes that are needed for understanding or producing speech are involved in operating with the rules.


**3. Generally applicable goals for bio-inspired design**

This section presents an analysis of the likelihood of satisfaction of selected goals with the use of the five methods for bio-inspired design, retrieved from literature and presented in the previous section. Five general goals are proposed that are deemed to encompass many of the requirements pertaining to design projects for which inspiration from nature is

From the observation of natural phenomena on a macro scale and / or a micro level, a potential solution to apply is sought to transfer to a human problem.

The components or systems involved in the phenomenon in question are identified in order to outline the biological solution in functional notation.

From the analysis of the biological solution in schematic notation, the fundamental principle of the solution is extracted.

In this case, reframing forces designers to think in terms of how humans might view the usefulness of the biological function being achieved.

Whereas search in the biological domain includes search through some finite space of documented biological solutions, the search may include defining new problems (this is much different than the solution search step in the problem-driven processes).

By analogy with the definition of the solution in schematic notation, the problem is outlined similarly. The aim is thus to establish a parallel between the systems and components of the biological solution and the problem.

Once the solution principle is established, it is transformed into a working principle of the technological concept that is needed. This activity will culminate in the embodiment of a bio-inspired solution of a technological product or system.

Table 5. Bio-solution in search of a problem method (adapted from Helms et al., 2009).

For the method presented in Table 4, the process of problem definition and searching for biological solutions is supported by elucidative techniques, suggestions and practical examples. The method presented in Table 5 supports an iterative formulation of the bio-

The application of bionic principles in a design project can be accomplished by following any of two opposing directions: finding a solution to a problem in nature, or looking for a problem for which a solution has been found in nature. The former approach starts with the identification of a problem (human applications, such as developing or improving products or services) or the need of a project, followed by looking for inspiration from nature or an analogy to foster a solution to the problem (a bionic solution proposal). This approach is well suited to designers seeking inspiration for the development of a particular product. The other approach is based on the observation of nature and its structures in order to collect useful information (bionic inspiration based solution) for human applications (design

This section presents an analysis of the likelihood of satisfaction of selected goals with the use of the five methods for bio-inspired design, retrieved from literature and presented in the previous section. Five general goals are proposed that are deemed to encompass many of the requirements pertaining to design projects for which inspiration from nature is

Phase Description

1. Biological solution identification

2. Define the biological solution

3. Principle extraction

4. Reframe the solution

> 5. Problem search

> 6. Problem definition

7. Principle application

inspired design principle.

problems to be sought).

**3. Generally applicable goals for bio-inspired design**

sought. The goals were selected based on their perceived level of importance and their perceived ubiquitous relevance across design projects, albeit translated into a number of requirements, specific to the problems at hand. Communication effectiveness, form optimization, multiple requirements satisfaction, organization effectiveness and paradigm innovation for improved functional performance are the goals considered.

Effectiveness of communication depends on the sharing of a language that may be based on a code, gestures, or on signal that is appropriate to the activity and context. For effective communication to accrue it is necessary that the message is clearly delivered and received in a timely fashion, without noise, and that it is relevant to the situation or event that is ongoing.

Optimizing the shape of an object or structure can result directly from the balanced satisfaction (with concessions on both sides - trade-offs) of several key requirements, such as the reduction of material and, or, size, or the satisfaction of greater stability, or reduced drag, depending on the targeted objectives. It is not always possible to achieve an optimal configuration, with maximization of all properties due to inherent conflicts that they sometimes impart (e.g. contradiction between low weight and high strength or high volume or stability). Thus, optimization requires that the configuration reached is the one that best addresses the contradictions and conflicts between the desired properties.

Nature is rife with effective solutions in order to enable, in a limited space, a system to perform various tasks or fulfil several functions. Compliance with multiple requirements reflects the achievement of several key points that are inherent to the problem at hand, aiming for viability and profitability of a small number of structures and elements that are to be used in performing more than one function. This simultaneous satisfaction opens the way for consideration of new objectives to add value and profitability to the designed product or system. Compliance with various targets, carried out by a limited set of features, structures or entities implies streamlining for functional efficiency, which will result in resource savings.

The effectiveness of organization depends on the coordination of multiple structures (which also includes communication) for the performance of activities with the need of differentiation. The coordination of multiple entities in joint activity may lead to more effective results than the performance of the activity separately by each entity, such as that "the whole is greater than the sum of its parts". An example of excellent coordination and effectiveness of the resulting organization can be inferred from observation of the natural system comprised of a pack of wolves. The group can hunt animals larger than the wolf, while a lone wolf may only hunt smaller animals or of a scale similar to his. The organization of the roles of each element within the pack is a pre-condition for achieving this result.

Finally, the fifth goal considered consists in achieving change in the conventional paradigm used to implement a feature, replacing it with an innovative paradigm. The latter may be proposed based on the observation of structures, behaviours and, or, processes of nature that enable improved performance of the function or feature. The features can be characterized by transformation of physical state, state association or state hierarchy, to name a few. This goal is deemed to represent one of the most commonly sought goals by designers inclined to use a bionic approach.

#### **3.1 Likelihood of achieving the goals selected by using bio-inspired methods**

Considering the five goals presented, the five methods under focus were analysed in terms of their perceived support offered to designers making use of them towards the satisfaction

Biologically Inspired Design: Methods and Validation 109

it is only implicit in the consideration of the analysis of ecosystems and natural social conditions. For the other goals at hand, this method proves to be applicable to support their satisfaction if the target is form optimization (especially given the nature of this iterative method, which favours systematic optimization) or innovation paradigm with regard to performance features. No support is perceived to attain the goal of communication

> Form optimization

Applicable with shortcomings

Applicable with shortcomings

Applicable with shortcomings

Applicable with shortcomings

Table 6. Analysis of perceived support provided by the five bio-inspired design methods

The bio-inspired design method shows gaps in the support offered to designers if the goal is to achieve optimal form, since the focus in this method is set on function. In some design processes supported by the procedures inherent to this method, the search for a biologically inspired solution to perform a given function could lead to considerations of form. However, the method does not provide procedures for optimization and does not explicitly consider form, or shape. The method is also deemed applicable in a satisfactory manner to problems where the targeted goal is either paradigm innovation for improved functional performance, or to satisfy multiple requirements, or a combination of both. However, it is not applicable to support the pursuance of the goals of effectiveness of either

Goals sought

Multiple requirements satisfaction

Not applicable

Applicable with shortcomings

Applicable with shortcomings

Applicable with shortcomings Organization effectiveness

Applicable with shortcomings

Applicable with shortcomings

Applicable Not applicable Applicable

Applicable Applicable

Applicable Not

Paradigm innovation for improved functional performance

Applicable

Applicable

applicable

effectiveness.

Bio-inspired design methods

Bio-mimicry (Junior et al., 2002)

Spiral design (Biomimicry Inst., 2007)

Bio-inspired design (Helms et al., 2009)

Aalborg (Colombo, 2007)

Bio-solution seeks problem (Helms et al., 2009)

Communication effectiveness

Not Applicable

Not Applicable

Not Applicable

Not Applicable

communication or organization.

selected in attaining five fundamental design goals.

Not Applicable Applicable

of each goal. In what concerns the effectiveness of organization, a method oriented from the solution to the problem (Aalborg) is considered applicable to support the satisfaction of this goal, demonstrating that there are a few gaps remaining in order to lead to the full satisfaction of this goal. None of the problem-oriented methods analyzed is considered fully adequate to achieve this goal.

With regard to satisfying multiple requirements, methods oriented from the solution to the problem show, from the analysis, gaps in support to achieve this goal. The methods providing guidance in implementing bionic projects in the contrary direction of analysis, are very heterogeneous. While the method of bio-mimicry offers no support for the pursuit of this goal, in the opposite extreme, with considerable support, is the method of bio-inspired design.

When considering the goal of form optimization, one is faced with a relatively homogeneous landscape, with the methods only offering partial support to attain this goal. The exception of the method of spiral design is highlighted, as it is considered significantly applicable in order to achieve this purpose (this method provides guidance in following the direction from problem to solution).

In what concerns the innovation of paradigm for improved functional performance, all analyzed methods provide satisfactory guidelines which can support the achievement of this purpose. This demonstrates that the primary approach that has been recommended for bionic design centres on the functionality. Moreover, except for individual cases, the remaining goals have been given a minor importance. Although the Aalborg and biomimicry method ratings were similar (except for organizational effectiveness), the steps of the latter are more detailed than the ones of the former, and there is a descriptive complementarity between both. The need to integrate validation activities in the bioinspired design processes is emphasized, as only a few of the methods (the spiral design and Aalborg design methods) entail some evaluation and iteration. The development and testing of improved methods, providing broad support to pursuing a large scope of design goals, with support for validation of the quality of results attained, is hence necessary. The results of the overall analysis are presented in Table 6.

The bio-mimicry design method is only deemed "applicable with shortcomings" with regard to attaining the goals of optimizing form and improving effectiveness of organization. For the first goal, the assessment derives from the absence of iteration in order to pursue optimization (observing the morphological structure is what is suggested in the method that may provide limited support to pursuing this goal). For the second goal, the assessment takes into account that the method supports no direct account of organizational aspects, but only does that indirectly through structural analysis. The evaluation also results in suggesting the applicability of the method to support the pursuance of the goal of paradigm innovation for increased functional performance, and on the other hand, enables suggesting its non-applicability if the goal is to achieve satisfaction of multiple requirements and communication effectiveness.

The spiral design method was granted the rating of "Applicable with shortcomings" with respect to the goal of satisfying multiple requirements. In this method, satisfaction of multiple requirements may take place according to their explanation in the initial specification, if natural models demonstrating the reunion of the functions and, or, qualities sought are analysed. However, the method does not explicitly consider a way to guide the quest to satisfy multiple requirements. The goal of organizational effectiveness receives the same evaluation, as the aspect of organization is not considered directly in this method, but

of each goal. In what concerns the effectiveness of organization, a method oriented from the solution to the problem (Aalborg) is considered applicable to support the satisfaction of this goal, demonstrating that there are a few gaps remaining in order to lead to the full satisfaction of this goal. None of the problem-oriented methods analyzed is considered fully

With regard to satisfying multiple requirements, methods oriented from the solution to the problem show, from the analysis, gaps in support to achieve this goal. The methods providing guidance in implementing bionic projects in the contrary direction of analysis, are very heterogeneous. While the method of bio-mimicry offers no support for the pursuit of this goal, in the opposite extreme, with considerable support, is the method of bio-inspired

When considering the goal of form optimization, one is faced with a relatively homogeneous landscape, with the methods only offering partial support to attain this goal. The exception of the method of spiral design is highlighted, as it is considered significantly applicable in order to achieve this purpose (this method provides guidance in following the

In what concerns the innovation of paradigm for improved functional performance, all analyzed methods provide satisfactory guidelines which can support the achievement of this purpose. This demonstrates that the primary approach that has been recommended for bionic design centres on the functionality. Moreover, except for individual cases, the remaining goals have been given a minor importance. Although the Aalborg and biomimicry method ratings were similar (except for organizational effectiveness), the steps of the latter are more detailed than the ones of the former, and there is a descriptive complementarity between both. The need to integrate validation activities in the bioinspired design processes is emphasized, as only a few of the methods (the spiral design and Aalborg design methods) entail some evaluation and iteration. The development and testing of improved methods, providing broad support to pursuing a large scope of design goals, with support for validation of the quality of results attained, is hence necessary. The results

The bio-mimicry design method is only deemed "applicable with shortcomings" with regard to attaining the goals of optimizing form and improving effectiveness of organization. For the first goal, the assessment derives from the absence of iteration in order to pursue optimization (observing the morphological structure is what is suggested in the method that may provide limited support to pursuing this goal). For the second goal, the assessment takes into account that the method supports no direct account of organizational aspects, but only does that indirectly through structural analysis. The evaluation also results in suggesting the applicability of the method to support the pursuance of the goal of paradigm innovation for increased functional performance, and on the other hand, enables suggesting its non-applicability if the goal is to achieve satisfaction of multiple requirements and

The spiral design method was granted the rating of "Applicable with shortcomings" with respect to the goal of satisfying multiple requirements. In this method, satisfaction of multiple requirements may take place according to their explanation in the initial specification, if natural models demonstrating the reunion of the functions and, or, qualities sought are analysed. However, the method does not explicitly consider a way to guide the quest to satisfy multiple requirements. The goal of organizational effectiveness receives the same evaluation, as the aspect of organization is not considered directly in this method, but

adequate to achieve this goal.

direction from problem to solution).

of the overall analysis are presented in Table 6.

communication effectiveness.

design.

it is only implicit in the consideration of the analysis of ecosystems and natural social conditions. For the other goals at hand, this method proves to be applicable to support their satisfaction if the target is form optimization (especially given the nature of this iterative method, which favours systematic optimization) or innovation paradigm with regard to performance features. No support is perceived to attain the goal of communication effectiveness.

Table 6. Analysis of perceived support provided by the five bio-inspired design methods selected in attaining five fundamental design goals.

The bio-inspired design method shows gaps in the support offered to designers if the goal is to achieve optimal form, since the focus in this method is set on function. In some design processes supported by the procedures inherent to this method, the search for a biologically inspired solution to perform a given function could lead to considerations of form. However, the method does not provide procedures for optimization and does not explicitly consider form, or shape. The method is also deemed applicable in a satisfactory manner to problems where the targeted goal is either paradigm innovation for improved functional performance, or to satisfy multiple requirements, or a combination of both. However, it is not applicable to support the pursuance of the goals of effectiveness of either communication or organization.

Biologically Inspired Design: Methods and Validation 111

Goal Validation procedures to evaluate goal accomplishment

by the sender (empirical verification).

intended for the bionic concept. Examples:

prototype testing.

multiple system elements.

and, or, technical schemes.

goals sought in bio-inspired design endeavours.

technology or kind of energy supplied.

Table 7. Specific procedures suggested for consideration of the processes of validation of

Validation is made according to the level of communication involved.

Active communication (synchronous process between a sender and a receiver) - effectiveness evaluated from the assessment of the overlap between the messages from the sender and what is

perceived by the receiver, which should conform to what is desired

Passive communication (triggered by observation) - the effectiveness may be evaluated by assessing the degree of the overlap between the meaning intended to be incorporated into the product or system by the designer and the readings of signification

made by users or observers (empirical verification).

Validation based on a comparative approach with regard to a conventional product with functionality that is similar to the one

Reducing material and weight - analysis from solid modeling. Stability - static analysis of mass centre (force vector modeling). Resistance for maximum capacity - finite element method and

Object storage - capacity, maximum capacity; quantification.

requirement. This is followed by checking if the resolution of conflicts between non-compatible properties was made with compromises

Validation based on objectively verifying, as much as possible, the level that has been reached for each property implicit in every

Validation based on the comparison between two or more systems performing the same function (including the proposed system), but with different methods of organization. Collect measures of the levels of operation effectiveness from the (real or simulated) systems (including the proposed system), such as execution time, energy expended, material resources expended, or resources generated.

The evidence of paradigm change depends on the type of paradigm involved. Consider these examples of two kinds of paradigm change: Paradigm change at the organizational level - could involve changing from a centralized model of decision making to a process of cooperative decision making distributed and performed by

Paradigm change at the technical level – could involve

The verification of the satisfaction of this goal may centre on a conceptual-analytical argument distinguishing between the existing and the new paradigm, possibly illustrated by descriptive imagery

fundamental changes in working principle, shape archetype, drive

established on every side of the requirements concerned.

Communication effectiveness

> Form optimization

Multiple requirements satisfaction

Organization effectiveness

Paradigm innovation for improved functional performance

In the Aalborg method, which provides guidance in the direction from the solution to the problem, the degree of applicability to the goals of form optimization and satisfaction of multiple conditions, was assigned as "Applicable with shortcomings". For the first goal, despite the focus on form, there is no effort to optimize. Secondly, because shape, structure and functional principles are considered in this method the, implementation of multiple principles of form and structure may result from the analysis but is not explicitly considered. For the goals of innovation of paradigm for improved performance of functions and for effectiveness of organization, this method is deemed applicable.

For the bio-solution in search of a problem method, which is directed from the solution to the problem, as this method focuses on extracting and implementing the solution principle form nature, both the aspects of optimizing the shape and satisfying multiple requirements, are bound to be sidelined at the expense of the functional principle. The evaluation of this method and the previous one only differ significantly on the applicability to provide support to the pursuance of the goal of organizational effectiveness, because in this method there is no focus on the organizational structure of the biological system centred upon. The method is deemed applicable to support attaining the goal of paradigm innovation for increased functional performance.

#### **4. Validation of goal satisfaction in the bio-inspired design process**

The analysis presented in the previous section identified goals where the methods considered were deemed to either offer no support, or only offer reduced support, towards their pursuance and satisfaction. Moreover, only two of the methods (the spiral design method and the Aalborg method) entail some evaluation procedures, albeit limited in scope. This leads to suggest the integration of validation activities in bionic design processes, in order to ascertain whether the desired goals might be met by the use of the concepts generated with the support of bionic design methods. The current section presents a proposed validation approach, summarily depicted on Table 7, based on considering specific validation procedures matching each of the five goals focused in the previous section.

#### **4.1 Bio-inspired design case requirements**

Five methods that are intended to support the generation of bio-inspired design concepts, retrieved from literature, were analysed in this chapter. Three of these methods shared a common direction of analysis, which departs from a given problem and seeks the proposal of solutions by gathering insight and inspiration form natural systems. This approach begins with the identification of a problem or the needs of a project, which is followed by looking for inspiration from nature or seeking an analogy with a natural solution to foster the emergence of a solution to the problem (a bionic solution proposal).

A bionic design project was carried out, following an approach combining three of the methods reviewed (Junior et al. 2002, Biomimicry Inst. 2007, Helms et al. 2009). The problem considered was the storage and the physical display to enable browsing of personal music collections, focusing on CDs and DVDs. The conduction of the design process led to seek inspiration form nature, having selected the spider web as a natural example that was the basis for the analogy of working principle established. The requirements established for the project and their corresponding goals are listed in Table 8. Moreover, environmental

In the Aalborg method, which provides guidance in the direction from the solution to the problem, the degree of applicability to the goals of form optimization and satisfaction of multiple conditions, was assigned as "Applicable with shortcomings". For the first goal, despite the focus on form, there is no effort to optimize. Secondly, because shape, structure and functional principles are considered in this method the, implementation of multiple principles of form and structure may result from the analysis but is not explicitly considered. For the goals of innovation of paradigm for improved performance of functions

For the bio-solution in search of a problem method, which is directed from the solution to the problem, as this method focuses on extracting and implementing the solution principle form nature, both the aspects of optimizing the shape and satisfying multiple requirements, are bound to be sidelined at the expense of the functional principle. The evaluation of this method and the previous one only differ significantly on the applicability to provide support to the pursuance of the goal of organizational effectiveness, because in this method there is no focus on the organizational structure of the biological system centred upon. The method is deemed applicable to support attaining the goal of paradigm innovation for

The analysis presented in the previous section identified goals where the methods considered were deemed to either offer no support, or only offer reduced support, towards their pursuance and satisfaction. Moreover, only two of the methods (the spiral design method and the Aalborg method) entail some evaluation procedures, albeit limited in scope. This leads to suggest the integration of validation activities in bionic design processes, in order to ascertain whether the desired goals might be met by the use of the concepts generated with the support of bionic design methods. The current section presents a proposed validation approach, summarily depicted on Table 7, based on considering specific validation procedures matching each of the five goals focused in the previous

Five methods that are intended to support the generation of bio-inspired design concepts, retrieved from literature, were analysed in this chapter. Three of these methods shared a common direction of analysis, which departs from a given problem and seeks the proposal of solutions by gathering insight and inspiration form natural systems. This approach begins with the identification of a problem or the needs of a project, which is followed by looking for inspiration from nature or seeking an analogy with a natural solution to foster the

A bionic design project was carried out, following an approach combining three of the methods reviewed (Junior et al. 2002, Biomimicry Inst. 2007, Helms et al. 2009). The problem considered was the storage and the physical display to enable browsing of personal music collections, focusing on CDs and DVDs. The conduction of the design process led to seek inspiration form nature, having selected the spider web as a natural example that was the basis for the analogy of working principle established. The requirements established for the project and their corresponding goals are listed in Table 8. Moreover, environmental

and for effectiveness of organization, this method is deemed applicable.

**4. Validation of goal satisfaction in the bio-inspired design process**

increased functional performance.

**4.1 Bio-inspired design case requirements**

emergence of a solution to the problem (a bionic solution proposal).

section.

Table 7. Specific procedures suggested for consideration of the processes of validation of goals sought in bio-inspired design endeavours.

Biologically Inspired Design: Methods and Validation 113

between 18 and 60, both male and female, and with diverse professional and knowledge

Fig. 1. Depiction of a conventional CD tower, and the two bio-inspired CD tower racks

Each respondent indicated which of the CD racks was personally more aesthetically pleasing and appealing, from 3 paired comparisons presented. The paired comparisons approach applied to this case of three objects enables 8 possibilities of response, two of which are incongruent, since no ranking of preference can be established out of them. Three out of the 85 respondents reported incongruent paired comparisons. Thus, the analysis of results was carried out for 82 responses. The results were analysed on the basis of the procedure for calculating the Kendall coefficient of concordance (Siegel & Castellan 1988). The average ranking obtained was bionic tower 1 (first place), bionic tower 2 (second place) and conventional tower (third place). This result is considered significant to represent the overall opinion of respondents to a confidence level of 99%. These results support the validation of the first requirement depicted in Table 8. Both the first and second bionic towers received the preference of respondents over the conventional tower, which supports the validation of the gains in terms of pleasantness and aesthetic appeal, for both versions of

In what concerns the second requirement that contributes to the goal of effective communication, validation was sought by means of a technique of anthropomorphizing products through the attribution of personality dimensions. In a first phase, a translation of the requirement into a product personality profile (Jordan 2002) was proposed. In the second phase of the process, a sample of specialized public (eight undergraduate Industrial Design students) assessed the personality profile of the three objects shown in Figure 1. In such, whether or not the message intended by the designer was transmitted to the public

The second requirement set in Table 8, was decomposed in a number of concepts to promote the matching process envisaged. This led to considering the attributes of modern, elegant, youthful, joyful, flexible and dynamic. Moreover the attributes consisting of lightweight and stable were also considered from the third and fourth requirements. The correspondence between product attributes intended by the designer to be perceived by the public and

designed: conventional tower(A), bionic tower 1 (B) and bionic tower 2 (C).

specialities.

the bio-inspired design.

could be verified.

concerns were expressed in terms of reduced environmental impact of materials, ease of maintenance and repair, as well as low weight of the product (and its package) for transportation. These requirements were dealt with in the design project, impinging on the selection of materials (selection of a bio-polymer and an organic elastomer) and on the design of the project (impinging on the goal of paradigm innovation for improved functional performance).

\*- Satisfaction of multiple requirements is implicit in the consideration of the several requirements; † - Conflicting requirements, requiring a trade-off.

Table 8. Listing of requirements set for the bio-inspired design case presented and their corresponding goals that were sought.

The validation processes carried out within the exemplified design case used are summarily described, and an overview of the evidence used and the results obtained is provided in the following sub-sections, considering the goals depicted in Table 7. In what concerns the goal of satisfaction of multiple requirements, a conflict was detected between the requirement of enhanced stability and lightness. This conflict was solved by means of an approach akin to TRIZ (Altshuller 1994), with the contradiction solved by change of state, in the second iteration of the design. Bionic tower 2 hence encompasses a reservoir in the basis which may be filled with water or sand for added stability, while lightness is still guaranteed, for the sake of environmental concerns, especially focusing on the production and distribution phases of the product's life-cycle.

#### **4.2 Validation of communication effectiveness**

The perception by the user of pleasantness and appeal, enabling the development of an aesthetic interest in the product (first requirement in Table 8) was validated through a questionnaire where, among other things, each of the two bionic CD towers was visually compared, with a conventional tower (Figure 1). The validation of this requirement is necessarily subjective, because the key issue that arises relates to the taste and sensitivity of each individual questioned. Respondents, answering by email, accounted to 85, aged

concerns were expressed in terms of reduced environmental impact of materials, ease of maintenance and repair, as well as low weight of the product (and its package) for transportation. These requirements were dealt with in the design project, impinging on the selection of materials (selection of a bio-polymer and an organic elastomer) and on the design of the project (impinging on the goal of paradigm innovation for improved

2. Sending a message of an avant-garde character, creative and effectiveness

4. Increased lightness compared to conventional solution† Form optimization

6. Storage with versatility of CDs, DVDs or books Organization

\*- Satisfaction of multiple requirements is implicit in the consideration of the several requirements;

The validation processes carried out within the exemplified design case used are summarily described, and an overview of the evidence used and the results obtained is provided in the following sub-sections, considering the goals depicted in Table 7. In what concerns the goal of satisfaction of multiple requirements, a conflict was detected between the requirement of enhanced stability and lightness. This conflict was solved by means of an approach akin to TRIZ (Altshuller 1994), with the contradiction solved by change of state, in the second iteration of the design. Bionic tower 2 hence encompasses a reservoir in the basis which may be filled with water or sand for added stability, while lightness is still guaranteed, for the sake of environmental concerns, especially focusing on the production and distribution

The perception by the user of pleasantness and appeal, enabling the development of an aesthetic interest in the product (first requirement in Table 8) was validated through a questionnaire where, among other things, each of the two bionic CD towers was visually compared, with a conventional tower (Figure 1). The validation of this requirement is necessarily subjective, because the key issue that arises relates to the taste and sensitivity of each individual questioned. Respondents, answering by email, accounted to 85, aged

Table 8. Listing of requirements set for the bio-inspired design case presented and their

1. Nice and appealing shape, enabling the user to develop an

youthful 3. Enhanced stability against a dynamic disturbance compared with a conventional solution†

5. Proper positioning of the title of the CDs, DVDs and books for enhanced readability

7. Enhanced gripping of objects compared with a conventional solution

† - Conflicting requirements, requiring a trade-off.

corresponding goals that were sought.

phases of the product's life-cycle.

**4.2 Validation of communication effectiveness**

Requirements Goals sought\*

effectiveness

Paradigm innovation for improved functional performance

aesthetic interest in product Communication

functional performance).

between 18 and 60, both male and female, and with diverse professional and knowledge specialities.

Fig. 1. Depiction of a conventional CD tower, and the two bio-inspired CD tower racks designed: conventional tower(A), bionic tower 1 (B) and bionic tower 2 (C).

Each respondent indicated which of the CD racks was personally more aesthetically pleasing and appealing, from 3 paired comparisons presented. The paired comparisons approach applied to this case of three objects enables 8 possibilities of response, two of which are incongruent, since no ranking of preference can be established out of them. Three out of the 85 respondents reported incongruent paired comparisons. Thus, the analysis of results was carried out for 82 responses. The results were analysed on the basis of the procedure for calculating the Kendall coefficient of concordance (Siegel & Castellan 1988). The average ranking obtained was bionic tower 1 (first place), bionic tower 2 (second place) and conventional tower (third place). This result is considered significant to represent the overall opinion of respondents to a confidence level of 99%. These results support the validation of the first requirement depicted in Table 8. Both the first and second bionic towers received the preference of respondents over the conventional tower, which supports the validation of the gains in terms of pleasantness and aesthetic appeal, for both versions of the bio-inspired design.

In what concerns the second requirement that contributes to the goal of effective communication, validation was sought by means of a technique of anthropomorphizing products through the attribution of personality dimensions. In a first phase, a translation of the requirement into a product personality profile (Jordan 2002) was proposed. In the second phase of the process, a sample of specialized public (eight undergraduate Industrial Design students) assessed the personality profile of the three objects shown in Figure 1. In such, whether or not the message intended by the designer was transmitted to the public could be verified.

The second requirement set in Table 8, was decomposed in a number of concepts to promote the matching process envisaged. This led to considering the attributes of modern, elegant, youthful, joyful, flexible and dynamic. Moreover the attributes consisting of lightweight and stable were also considered from the third and fourth requirements. The correspondence between product attributes intended by the designer to be perceived by the public and

Biologically Inspired Design: Methods and Validation 115

According to the findings obtained, the communication of a message of young spirit, dynamism and joyfulness were validated. Tower C (bionic tower 2) is the one which, according to the survey, more effectively conveys the desired messages, is considered the most dynamic, the most rebellious, most joyful and, together with tower B (bionic tower 1), most kind and most liberal. Regarding the transmission of the message of lightness, the personality profile related (simple - complex) did not translate so well the associated requirement. This might have led respondents to identify tower A (conventional) as the simplest, and therefore, according to the tenuous association, the lightest of the three. Interpretative meanings vary from person to person. The absence of actual experience of use of the towers on the part of respondents, who just exercised visual perception, may have also influenced and contributed to vagueness and lack of agreement among the

The results concerning requirements contributing to the satisfaction of the goal of form optimization are shown in Table 10 (enhanced stability – according to force vector analysis), Table 11 (increased lightness – solid modelling analysis), Figure 2 (enhanced readability of CD titles – graphical depiction) and Figure 2 (scheme illustrating analytical stability modelling). For the first of the three requirements concerned by this goal, bionic tower 2 ranks in first place, while for the second requirement, bionic tower 1 is clearly the lightest, while for the last of the three requirements both bionic towers achieve a tie ahead of the conventional tower. The results support validation of the achievement of the goal sought of form optimization, albeit both bionic towers are deemed equivalent in this

capacity 49,96 N 49,59 N 74 N

Table 10. Comparison of results for the maximum lateral disturbance tolerated without loss

At medium capacity 35,11 N 29,57 N 56,34 N

Total mass Conventional tower Bionic tower 1 Bionic tower 2

Transport mode 11,049 Kg 4,763 Kg 8,087 Kg

Use mode 11,049 Kg 4,763 Kg 17,087 Kg

Table 11. Comparison of mass data among the three objects.

Conventional tower Bionic tower 1 Bionic tower 2

respondents.

respect.

Maximum lateral disturbance to maintain stability

At maximum

of stability in the three concepts.

**4.3 Validation of form optimization**

product personality dimensions (Jordan 2002) are shown in Table 9. The outcome of analysis on the respondents assessment of the personality profiles is also shown, based on evaluation of Kendall's coefficient of concordance (Siegel & Castellan 1988).

For every personality pair, analysis was performed as exemplified for the pair energetic – non energetic energy, the average ranking of the panel of respondents (with a significance of 99%, given by the assessment of Kendall 's coefficient) resulted in the following rank order: 1st C, 2nd B, 3rd A. As a conclusion to this result, it is understandable that tower C (bionic tower 2) is considered more energetic than the tower B (bionic tower 1), and that tower C (conventional tower) is considered less energetic than tower B. This means that tower C is deemed the least energetic of the three towers and that C is the tower that emerges as the most dynamic and the less dynamic, thus validating this communication requirement.

Table 9. Analysis of the results of the survey on the personality profile of the towers for CD and DVD storage and verification of messages perceived from observation of the objects by the pannel of undergraduate industrial design students.

product personality dimensions (Jordan 2002) are shown in Table 9. The outcome of analysis on the respondents assessment of the personality profiles is also shown, based on

For every personality pair, analysis was performed as exemplified for the pair energetic – non energetic energy, the average ranking of the panel of respondents (with a significance of 99%, given by the assessment of Kendall 's coefficient) resulted in the following rank order: 1st C, 2nd B, 3rd A. As a conclusion to this result, it is understandable that tower C (bionic tower 2) is considered more energetic than the tower B (bionic tower 1), and that tower C (conventional tower) is considered less energetic than tower B. This means that tower C is deemed the least energetic of the three towers and that C is the tower that emerges as the most dynamic and the less dynamic, thus validating this communication requirement.

> Significance level of Kendall's coefficient of concordance

Complex A – B – C 99% Tower A is considered

Violent B and C – A Not significant Sample did not reveal

Excessive A – B – C Not significant Sample did not reveal

Conformist C – B – A 99% Tower C is the most

Pessimistic B – C – A Not significant Sample did not reveal

Unkind B and C - A 95% Towers B and C are the

Inflexible C – B – A Not significant Sample did not reveal

Unenergetic C – B – A 99% Tower C is the most

Table 9. Analysis of the results of the survey on the personality profile of the towers for CD and DVD storage and verification of messages perceived from observation of the objects by

Unstable B – A – C Not significant Sample did not reveal

B and C - A 99% Towers B and C are the

C – B – A 99% Tower C is the most light-

Conclusion

agreement

most simple (lightweight)

agreement

agreement

most liberal (youthful)

rebellious (youthful)

agreement

hearted (joyful)

most kind (joyful)

agreement

energetic (dynamic)

agreement

evaluation of Kendall's coefficient of concordance (Siegel & Castellan 1988).

Average ranking 1st- 2nd – 3rd

Modern Bright – Dim B – A – C Not significant Sample did not reveal

Designer's message

Elegant

Youthful spirit

Joyful

Lightweight Simple –

Personality profile

Gentle –

Moderate -

Liberal – Authoritarian

Rebel –

Optimistic –

Light-hearted – Seriousminded

Kind –

the pannel of undergraduate industrial design students.

Flexible Flexible –

Dynamic Energetic –

Stable Stable -

According to the findings obtained, the communication of a message of young spirit, dynamism and joyfulness were validated. Tower C (bionic tower 2) is the one which, according to the survey, more effectively conveys the desired messages, is considered the most dynamic, the most rebellious, most joyful and, together with tower B (bionic tower 1), most kind and most liberal. Regarding the transmission of the message of lightness, the personality profile related (simple - complex) did not translate so well the associated requirement. This might have led respondents to identify tower A (conventional) as the simplest, and therefore, according to the tenuous association, the lightest of the three. Interpretative meanings vary from person to person. The absence of actual experience of use of the towers on the part of respondents, who just exercised visual perception, may have also influenced and contributed to vagueness and lack of agreement among the respondents.


**Fundamentals of Multi-User MIMO Communication**

Chapter 7 will be dedicated to user selection based error probability of MIMO detector in multi-user MIMO system. Low complexity greedy user selection approach with suboptimal

This book focuses on multi-user MIMO communication and covers a collection of the major topics and issues in multi-user MIMO systems. « Recent Trends in Multi-user MIMO Com‐ munications » provides a tutorial overview of the latest technologies and research keys re‐

The target audience of this book comprises researchers, Ph.D. students, academicians and pro‐ fessionals working in the field of Signal Processing and Multiple Antenna Systems. It provides an interdisciplinary guide that can help the reader about the multi-user MIMO communication.

**Maha BEN ZID**

France

National School of Engineering of Le Mans (ENSIM)

Lattice Reduction (LR)-based detector will be studied.

lated to multi-user communication.

VIII Preface

**Chapter 1**

**Provisional chapter**

**Multi User MIMO Communication: Basic Aspects,**

**Multi User MIMO Communication: Basic Aspects,**

The explosive growth of Multiple Input Multiple Output (MIMO) systems has permitted for high data rate and a wide variety of applications. Some of the technologies which rely on these systems are IEEE 802.11, Third Generation (3G) and Long Term Evolution (LTE) ones. Recent advances in wireless communication systems have contributed to the design of multi-user scenarios with MIMO communication. These communication systems are referred as multi-user MIMOs. Such systems are intended for the development of new generations of wireless mobile radio systems for future cellular radio standards. This chapter provides an insight into multi-user MIMO systems. We firstly present some of the main aspects of the MIMO communication. We introduce the basic concepts of MIMO communication as well as MIMO channel modeling. Thereafter, we evaluate the MIMO system performances. Then, we concentrate our analysis on the multi-user MIMO systems and we provide the reader a conceptual understanding with the multi-user MIMO technology. To do so, we present the communication system model for such emerging technology and we give some examples which describe the recent advances for multi-user MIMO systems. Finally, we introduce linear precoding techniques which could be exploited in multi-user MIMO systems in order

The main historical events which make the MIMO systems [2][3] are summarized as follows:

• In 1984, Jack Winters at Bell Laboratories wrote a patent on wireless communications using multiple antennas. Jack Winters in [4] presented a study of the fundamental limits

> ©2012 Ben Zid, Raoof, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Maha and Kosai; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

on the data rate of multiple antenna systems in a Rayleigh fading environment.

**Benefits and Challenges**

**Benefits and Challenges**

Ben Zid Maha and Raoof Kosai

http://dx.doi.org/10.5772/57133

to suppress inter-user interference.

**2. MIMO communication**

**2.1. An historical overview**

10.5772/57133

**1. Introduction**

Ben Zid Maha, Raoof Kosai

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**


**1. Introduction**

Tumor angiogenesis, the formation of new blood vessels in solid tumors, plays an important role in tumor cell survival, growth, and metastasis [1]. A major driving force of tumor angiogenesis is the signaling pathway involving vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) [2]. Several angiogenesis inhibitors, including antibodies and small molecule compounds targeting the VEGF/VEGFR signaling pathway, have been approved by the Food and Drug Administration (FDA), and used for the treatment of many different types of cancers [3]. Besides cancer treatment, VEGF/VEGFR inhibitors, including antibody fragments, aptamers, and VEGF-Traps were also approved and used for the treatment of ocular diseases caused by pathological angiogenesis [4–8]. VEGF/VEGFR blockade can inhibit VEGF-driven tumor angiogenesis, and the regression of tumor vessels is dependent on the VEGF signaling pathway. However, VEGF inhibitors alone are not capable of destroying all tumor blood vessels. In addition, preclinical studies indicate that VEGF inhibitors alone resulted in an increasingly aggressive and invasive pattern of tumors [9]. Some cancer patients are eventually refractory to anti-VEGF therapy,

**Citation:** Yeom, D.-H.; Lee, Y.-S.; Ryu, I.; Lee, S.; Sung, B.; Lee, H.-B.; Kim, D.; Ahn, J.-H.; Ha, E.; Choi, Y.-S.; et al. ABL001, a Bispecific Antibody Targeting VEGF and DLL4, with Chemotherapy, Synergistically Inhibits Tumor Progression in Xenograft Models. *Int. J. Mol. Sci.* **2021**, *22*, 241. https://doi.org/ 10.3390/ijms22010241

Received: 7 December 2020 Accepted: 25 December 2020 Published: 29 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/ licenses/by/4.0/).

hence, next-generation angiogenesis inhibitors are being sought to augment the effects of VEGF inhibitors [10–12].

The DLL4/Notch signaling pathway can be a promising target of the next angiogenesis inhibitors, as this pathway regulates tumor angiogenesis with a different mechanism of action compared to that of the VEGF inhibitors [13–15]. Several preclinical xenograft studies have demonstrated that DLL4/Notch blockade inhibited tumor progression by promoting hyperproliferation of endothelial cells, which resulted in an increase in vascular density and a decrease in functional tumor vasculature [14–20]. DLL4/Notch inhibition is also known to reduce the number of cancer stem cells (CSCs), which are an important cancer cell population responsible for malignancy [21]. ABL001 is a bispecific antibody that simultaneously targets both DLL4 and VEGF, by linking each C-terminal of an anti-VEGF antibody (bevacizumab-similar) with a DLL4-binding single-chain Fv (scFv) [22,23]. In previous studies, ABL001 has demonstrated anti-cancer effects with higher potency in several human cancer xenograft models compared to that shown by the VEGF-targeting antibody (bevacizumab-similar) and the DLL4-targeting monoclonal antibody alone [23,24].

The safety and tolerability of ABL001 in cancer patients are now being evaluated in a phase 1 dose escalation study. The study was designed in a classical 3+3 dose-escalation schema where ABL001 is administered by IV across nine dose cohorts ranging from 0.3, 1, 2.5, 5, 7.5, 10, 12.5, 15, to 17.5 mg/kg biweekly [25]. No dose-limiting toxicity (DLT) was observed during the final cohort dose (17.5 mg/kg), and the maximum tolerated dose (MTD) was not reached. The most common treatment-related adverse events (AEs) (including all dose levels and all grades) were hypertension, anemia, anorexia, general weakness, and headache. However, they were well managed for all cohorts. Although the current phase 1 trial of monotherapy of ABL001 is ongoing, further clinical studies should be performed in combination with chemotherapy after the selection of optimal anti-cancer agents and cancer types. Since angiogenesis inhibitors target tumor endothelial cells, most VEGF/VEGFR blocking agents demonstrate clinical benefits for cancer patients when combined with chemotherapy [3]. Two different mechanisms of action of the combination therapy could provide synergistic anti-cancer efficacy for cancer patients. First, the combination therapy can destroy two separate components of tumors, tumor cells and tumor endothelial cells [26,27]. Second, the tumor vessel normalization by angiogenesis inhibitors enhances the delivery of cytotoxic anti-cancer agents [28,29]. However, the effects of a combination of ABL001 with chemotherapy on tumors and tumor blood vessels have not been fully studied. In this report, the in vivo anti-cancer effects of ABL001 with chemotherapy were evaluated in human gastric and colon cancer xenograft models and were compared to each monotherapy alone.

#### **2. Results**

#### *2.1. Suppression of Tumor Progression in Various Cancer Xenograft Models by ABL001*

To confirm the effects of ABL001 on tumor progression and to select the appropriate xenograft models for testing a combination treatment of ABL001 with chemotherapy, we evaluated the anti-cancer effects of ABL001 using several human gastric cancer (NUGC-3, MKN45, and SNU16 for mABL001, and GAPF006 for ABL001) xenograft models (Figure 1A), and human colon cancer (Colo205, WiDr, SW48, and SW620 for mABL001) xenograft models (Figure 1B). In the case of general xenograft models using human cancer cell lines, we used the mouse surrogate version of ABL001 (mABL001: binding to human VEGF and mouse DLL4) for the studies, as DLL4 is expressed by mouse endothelial cells involving tumor angiogenesis in tumor xenografts [23]. However, we used ABL001 in a patient-derived xenograft (PDX) model using GAPF006, which mimics the human tumor microenvironment from patients. Both bispecific antibodies, mABL001 and ABL001, inhibited tumor progression in the tested xenograft models at doses ranging from 1 to 6.5 mg/kg (Figure 1). The anti-cancer effects of mABL001 or ABL001 monotherapy were calculated as %TGI ranging from 27.4% to 57.2%, depending on the doses of mABL001 or ABL001 and cancer cell lines in xenograft models (Table 1). We focused on the dose level of ABL001

showing %TGI50 (50% tumor growth inhibition ratio) in each xenograft model because the dose of ABL001 and the xenograft model would be used for the combination therapy with paclitaxel or irinotecan. Based on the results from the dose range-finding studies, we selected GAPF006 gastric PDX, and SW48 or SW620 colon cancer xenograft models to address the efficacy of the combination treatment.

**Figure 1.** ABL001 strongly inhibited tumor progression of various human gastric and colon cancer xenograft models. Tumor size was measured twice per week and compared between vehicle (closed circle) and ABL001 (closed triangle) in human gastric cancer (NUGC-3, MKN45, SNU16 for mABL001, and human patient-derived gastric cancer GAPF006 for ABL001) xenograft model (**A**) and human colon cancer (Colo205, WiDr, SW48, SW620 for mABL001) xenograft model (**B**). ABL001 treatment significantly delayed tumor progression in different cancer xenograft models compared to control group of vehicle treatment. Error bars: mean ± SEM.

**Table 1.** Summarized information of animal studies using human gastric and colon cancer xenograft models.

GAPF006, gastric patient-derived xenograft model, %TGI = tumor growth inhibition, *p* value: Student's *t*-test.

#### *2.2. Synergistic Suppression on Tumor Progression by Combination Therapy*

To determine whether the combination treatment of ABL001 with chemotherapy suppressed tumor progression with a higher strength as compared to that of each monotherapy, we evaluated the anti-cancer effects of the combination therapy using xenograft models compared to ABL001 or chemotherapy alone (Figure 2). In this study, we tested paclitaxel as chemotherapy in combination with ABL001 in gastric GAPF006 PDX (human gastric origin) xenograft, and irinotecan with mABL001 in SW48 or SW620 human colon tumor xenografts. In the gastric PDX model, the combination of paclitaxel and ABL001 demonstrated the most potent inhibition of tumor progression (74.75% TGI compared to 40.33% TGI in the paclitaxel-treated group and 46.20% TGI in the ABL001-treated group) (Figure 2A). Similarly, the combination of irinotecan with mABL001 suppressed tumor progression of SW48 and SW620 human colon cancer xenografts more potently compared to that by irinotecan or mABL001 alone (Figure 2B,C). At the endpoint of the SW48 xenograft study, the combination of irinotecan and mABL001 demonstrated 77.7% TGI, which was significantly different from the %TGI of the vehicle (*p* < 0.0001) group and irinotecan (*p* < 0.005) or mABL001 alone (*p* < 0.05) (Figure 2B). In the case of the SW620 xenograft model (human colon cancer), the combination treatment of irinotecan and mABL001 also exhibited the most potent anti-cancer effect (94.47% TGI) on tumor progression in the SW620 xenograft (Figure 2C).

**Figure 2.** ABL001 in combination with chemotherapy with paclitaxel or irinotecan synergistically inhibited tumor progression in human gastric PDX and colon cancer xenograft models. In GAPF006 human gastric PDX model (**A**), mice were treated with vehicle (closed circle, black), paclitaxel alone (closed rectangle, green), ABL001 (closed triangle, blue), or a combination of ABL001 and paclitaxel (closed reverse triangle, red). Compared to vehicle, each treatment group inhibited tumor progression (40.33% TGI in paclitaxel, 46.20% TGI in ABL001, and 74.75% TGI in the combination treatment). In the studies using SW48 (**B**) and SW620 (**C**) colon cancer xenograft models, mice were treated with vehicle (closed circle, black), irinotecan alone (closed rectangle, green), mABL001 (closed triangle, blue), or a combination of mABL001 and irinotecan (closed reverse triangle, red). In the case of both colon cancer xenograft models, the combination treatment of mABL001 and irinotecan showed the most potent effects on tumor progression (77.7% TGI in SW48 and 94.47% TGI in SW620 xenograft models). Each line represents the average tumor size (mm3) of each treatment group ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 by Tukey's test.

#### *2.3. More Potent Regression of Tumor Vessels by Combination Therapy*

In order to evaluate the effects of the combination therapy on tumor blood vessels in xenograft models, the tumor vessels of SW620 tumor sections were analyzed using immunohistochemical staining for CD31 and VEGFR-2. Fluorescence microscopy images revealed that CD31-positive staining was localized in the vascular endothelial cells in the tumors (Figure 3A). The tumor vessel densities positive for CD31 in SW620 tumors treated with vehicle, irinotecan, mABL001, and combination were 0.71 ± 0.05%, 0.48 ± 0.03%, 0.36 ± 0.03%, and 0.18 ± 0.01%, respectively (Figure 3B). The percentage of positive area for CD31 in the combination was significantly lower than that of irinotecan or mABL001 alone. The area density of CD31-positive vessels in irinotecan-treated tumors was decreased by 32.4% and the density in mABL001-treated tumors was decreased by 49.3%, compared to the vehicle-treated group. However, the density of CD31-positive tumor vessels in the

combination treatment decreased by 74.6% compared to the vehicle group (Figure 3B). VEGFR-2 was also strongly expressed on the endothelial cell membrane and cytoplasm in SW620 tumors (Figure 3A). The area densities of VEGFR-2-positive tumor vessels in the four groups were 0.65 ± 0.06%, 0.43 ± 0.04%, 0.23 ± 0.02%, and 0.13 ± 0.02%, respectively (Figure 3C). Compared to the vehicle-treated group, VEGFR-2-positive tumor vessels were reduced by 33.8% in the irinotecan-treated group, by 64.6% in the mABL001-treated group, and by 80% in the combination treatment group (Figure 3C). Based on the comparison of relative reduced levels between CD31-positive vessels with VEGFR-2-positive vessels in each tumor, VEGFR-2 expression was more reduced in tumor blood vessels compared to CD31 expression after VEGF blockade, mABL001 treatment, or the combination treatment (Figure 3B,C).

**Figure 3.** Combination therapy more potently regressed tumor blood vessels in SW620 xenograft model. Representative immunofluorescence images (**A**) show the tumor vasculature in SW620 tumor tissues stained for CD31, a generally conserved endothelial cell marker (green) and VEGFR-2 (red) with DAPI (blue). Most tumor blood vessels in vehicle group were stained and colocalized with both markers, CD31 and VEGFR-2. The area densities of CD31 (**B**) and VEGFR-2 (**C**) positive vessels were measured in each group. After irinotecan treatment, CD31 or VEGFR-2 positive tumor blood vessels were slightly regressed compared to vehicle treatment. However, after mABL001 or the combination treatment of mABL001 and irinotecan, CD31 and VEGFR-2 positive tumor vessels were significantly reduced (**B**,**C**). VEGFR-2 expression reduced more rapidly on tumor vessels. Scale bar indicates 200 μm. Error bars: mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001 by Kruskal–Wallis test.

#### *2.4. Decrease of DLL4 Expression on Tumor Vessels by Combination Therapy*

DLL4 is expressed by tumor endothelial cells to regulate tumor angiogenesis, and by some tumor cells to maintain cancer stemness [14,15,21]. To address whether treatment with irinotecan, mABL001, or their combination affects DLL4 expression in tumors of xenograft models, DLL4 expression was examined using immunohistochemical staining using SW620 tumor sections from each group (Figure 4). DLL4 was mainly expressed on tumor blood vessels rather than tumor cells in this xenograft tumor and colocalized with CD31-positive tumor vessels (Figure 4A). The area densities of DLL4-positive tumor vessels were 0.40 ± 0.03% in vehicle, 0.24 ± 0.03% in irinotecan, 0.11 ± 0.02% in mABL001, and 0.05 ± 0.01% in the combination treatment group, respectively (Figure 4B). DLL4-positive tumor vessels were significantly reduced in the combination group compared to other groups. Compared to the vehicle group, DLL4-positive tumor vessels were reduced by 40% in the irinotecan group, by 72.5% in the mABL001 group, and by 87.5% in the combination group (Figure 4B). Similar to VEGFR-2 expression in tumor vessels, DLL4 expression was markedly reduced in tumor vessels compared to CD31 after treatment with mABL001 or the combination, rather than treatment with irinotecan alone (Figure 4B). Such a rapid reduction of DLL4 expression after mABL001 caused some tumor vessels to be stained only by CD31 but not by DLL4 (arrows in Figure 4A).

**Figure 4.** ABL001 significantly reduced DLL4 expression in tumor blood vessels. Representative immunofluorescence images (**A**) indicate the tumor vasculature in SW620 tumor tissues stained for CD31 (green) and DLL4 (red). The bottom figures (**A**) are magnified images of the dotted region of the combination treatment of mABL001 and irinotecan. The left image was shown only by red channel, whereas the right one was shown by merged channels (red and green). Similar to VEGFR-2, DLL4 was stained and colocalized on CD31 positive tumor blood vessels. The area density of DLL4 (**B**) positive vessels was measured in tumors of each group. Compared to vehicle or irinotecan treatment, DLL4 positive tumor vessels were significantly reduced in tumors after mABL001 or the combination treatment. Some tumor vessels were stained only for CD31 but not for DLL4, after mABL001 or the combination treatment group (arrows and dotted box in A). Scale bar indicates 50 μm in the bottom two images and 100 μm in the other images. Error bars: mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001 by Kruskal–Wallis test.

#### *2.5. Increase of Tumor Apoptosis by Combination Therapy*

Since the combination treatment of mABL001 with irinotecan showed more potent anti-cancer effects on tumor progression and anti-angiogenic effects on tumor vessels, the effects of the combination therapy on tumor cells were analyzed by immunohistochemical staining for activated caspase-3, an apoptotic cell marker. Immunofluorescence imaging revealed that activated caspase-3 was largely stained in the tumor cell nuclei rather than in the tumor endothelial cell nuclei in the tumor sections (Figure 5A). The area densities of activated caspase-3/DAPI positive cells were 5.16 ± 0.74% in the vehicle-treated group, 7.92 ± 1.05% in the irinotecan-treated group, 8.92 ± 1.65% in mABL001-treated group, and 10.87 ± 1.78% in the combination group (Figure 5B). The level of apoptotic tumor cells was significantly increased in the tumor sections after the combination treatment compared with the other groups. Such a potent increase in tumor cell apoptosis by the combination treatment might be due to direct cytotoxic effects of irinotecan against highly proliferating tumor cells together with the anti-angiogenic effects of mABL001, a bispecific antibody binding against dual antigens, VEGF, and mouse DLL4. The results suggest that the combination treatment of ABL001 with chemotherapy might provide better clinical benefits for cancer patients in clinical trials than ABL001 monotherapy.

**Figure 5.** Combination therapy markedly increased apoptotic tumor cells in SW620 xenograft model. Representative immunofluorescence images (**A**) reveal apoptotic cells stained for activated caspase-3 (green) with DAPI (blue) in SW620 tumor tissues. The area densities of activated caspase-3-positive apoptotic cells were measured in each group (**B**). Apoptotic cells in tumors were marginally increased after irinotecan or mABL001 treatment, but the increase was not significant compared to vehicle treatment. However, the combination treatment of mABL001 and irinotecan markedly increased the apoptotic cell population in tumors. Scale bar indicates 50 μm. Error bars: mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001 by Kruskal–Wallis test.

#### **3. Discussion**

ABL001 (NOV1501/TR009), a bispecific antibody targeting VEGF and DLL4, is being developed as an anti-angiogenic cancer therapeutic that strengthens the effects of VEGF inhibitors and eventually overcomes resistance to anti-VEGF therapy [16,19,20,23]. ABL001 demonstrated more potent anti-angiogenic and anti-cancer effects in vitro and in vivo, as compared to the VEGF-targeting or the DLL4-targeting monoclonal antibodies alone, in various assay systems [23,24]. Based on the overall results of preclinical studies, the safety and tolerability of ABL001 are currently being tested with cancer patients previously treated heavily with chemotherapy or targeted therapy [25]. Other approved anti-angiogenic antibody therapeutics including bevacizumab, an antagonist of the VEGF ligand (VEGF-A: Avastin®), and ramucirumab, an antagonist of the VEGF receptor (VEGFR-2: Cyramza®), are generally used in a combination regimen with chemotherapy to treat cancer patients, providing more efficacious therapeutic options for cancer patients [3,30]. Anti-VEGF therapy is known to normalize tumor blood vessels, leading to a more efficient delivery of cytotoxic anti-cancer agents into tumor tissues [28,29], hence, most anti-VEGF therapy are used in the clinic in combination with chemotherapy [3,30]. Based upon the rationale mentioned above, newly developing VEGF/VEGFR inhibitors of monoclonal or bispecific

antibodies, small molecule compounds, aptamers, and VEGF-Traps, have been evaluated synergistic anti-cancer effects with chemotherapy in various preclinical models before entering clinical trials [27,31,32].

Not only VEGF but DLL4 is also known to impair efficient delivery of anti-cancer drugs and to enhance chemoresistance in pancreatic cancer model due to induction of defective tumor angiogenesis [33]. However, little is known about the effects of a combination of ABL001, targeting dual antigens VEGF and DLL4, and chemotherapy on tumor vessels and tumor cells in xenograft models compared to each monotherapy alone. In this study, we evaluated the anti-angiogenic and anti-cancer effects of the combination treatment of ABL001 with paclitaxel or irinotecan in human gastric and colon cancer xenograft models.

The combination treatment of ABL001 with paclitaxel or irinotecan demonstrated more potent inhibition of tumor progression in these xenograft models, which is consistent with the previous report of the study collaborator [24]. Such potent anti-cancer effects of the combination therapy might be related to more significantly regressed tumor blood vessels, as compared to monotherapy with ABL001 or chemotherapy alone. Eventually, these anti-angiogenic and anti-cancer effects increased the apoptotic tumor status in the tumors post the combination treatment of ABL001 and chemotherapy. The underlying molecular mechanisms of action of the potent anti-cancer effects of ABL001 with chemotherapy might be due to the optimal combination effects of cytotoxic activity on tumor cells by paclitaxel or irinotecan together with more potent anti-angiogenic activity on tumor endothelial cells by ABL001, a VEGF, and DLL4 dual inhibitor. Moreover, because the VEGF-binding part of ABL001 is composed of the same IgG backbone and sequence as bevacizumab, ABL001 may have similar activity and function as bevacizumab in tumor vessels, resulting in a more effective delivery of anti-cancer agents, such as paclitaxel or irinotecan.

Based on the results of immunohistochemical analysis of tumor blood vessels, the expression levels of VEGFR-2 and DLL4, dual targets of ABL001, were markedly reduced in tumor endothelial cells after ABL001 treatment compared to that of CD31, a conventional endothelial cell marker. These findings are consistent with the previous results that VEGF blockade downregulates the levels of its receptor, VEGFR-2, and of DLL4 on endothelial cells [34,35]. Therefore, these results strongly support that the VEGF/VEGFR signaling pathway interacts with the DLL4/Notch signaling pathway in the tumor vasculature [35].

In addition to the cytotoxic anti-cancer agents, tumor vessel normalization by anti-VEGF therapy is also able to provide a better infiltration of immune cells, including cytotoxic T cells, into tumor tissues [36]. These reports suggest that anti-VEGF therapy can be the best option for combination therapy with immune checkpoint inhibitors for non-responsive cancer patients due to the lack of immune cells in the tumors, which are so-called 'cold tumors' or 'non-inflamed tumors'. Indeed, a number of clinical studies for combination trials using anti-VEGF therapy with immune checkpoint inhibitors are ongoing for various cancer types [37]. During the past two years, 114 new combination regimens of VEGF and immune checkpoint inhibitors entered into clinical studies [38,39]. Among a large number of clinical studies, the FDA has approved several combination regimens of VEGF and immune checkpoint inhibitors, such as atezolizumab (an antagonist of PD-L1, Tecentriq®) plus bevacizumab with carboplatin and paclitaxel for the treatment of non-small cell lung cancer (NSCLC), avelumab (an antagonist of PD-L1, Bavencio®) plus axitinib (AG013736, a small molecule inhibitor of VEGFR tyrosine kinase, Inlyta®), and pembrolizumab (an antagonist of PD-1, Keytruda®) plus axitinib for the treatment of advanced renal carcinoma [40–42]. Recently, another combination regimen of atezolizumab plus bevacizumab was approved for the treatment hepatocellular carcinoma (HCC) as a first-line therapeutic option [43]. In this point of view, the results obtained in the current study imply that ABL001 may be another promising partner for combination therapy with immune checkpoint inhibitors, through the facilitation of immune cell infiltration via dual blockade of VEGF and DLL4 [28,29,33].

Currently, ABL001 is being tested for its safety, tolerability, and efficacy in phase 1 clinical studies with heavily pre-treated metastatic cancer patients. ABL001 has been well tolerated and no DLT is observed during dose escalation up to the final cohort, with manageable adverse effects generally exhibited by anti-cancer antibody therapeutics [25]. After the current dose escalation study of ABL001, further clinical development is scheduled to evaluate the efficacy of ABL001 in combination with chemotherapy. In conclusion, the results of this study provide important information for the clinical study design and plan for the combination treatment of ABL001 with chemotherapy.