Figure 1 The workflow of CAR development from a hybridoma

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Figure 1 The workflow of CAR development from a hybridoma Figure 1 The workflow of CAR development from a hybridoma. (A) The visual depiction of a second-generation CAR design. ... Figure 1 The workflow of CAR development from a hybridoma. (A) The visual depiction of a second-generation CAR design. (B) A simplified roadmap of CAR development and assessment with the focus of the article indicated by the green part of the diagram. (C) Validation of target antigen expression in MAVER-1 (Igλ+, dark gray) and BL-41 (Igκ+, light gray) cell lines with the indicates antibodies. The upper panel was stained with commercial antibodies, and lower was done using the hybridoma supernatants in duplicates at the indicated dilutions. Representative data of two independent experiments are shown. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Antibody Therapeutics.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Antibody Ther, Volume 2, Issue 2, 31 May 2019, Pages 56–63, https://doi.org/10.1093/abt/tbz007 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2 Investigating the hybridoma by 5′-RACE Figure 2 Investigating the hybridoma by 5′-RACE. (A) The depiction of 5′-RACE step by step protocol. (B) List of ... Figure 2 Investigating the hybridoma by 5′-RACE. (A) The depiction of 5′-RACE step by step protocol. (B) List of primers used in this study and references to already published ones. (C) Acquired gel pictures throughout the 5′-RACE protocol, each kappa and lambda hybridomas were divided into two and the protocol was followed in two replicates (e.g. named as K#1 and K#2). The top picture shows the result of the cDNA synthesis, the middle pictures the first PCR results of dC tailed samples and the bottom pictures the result of the second PCR performed with the first PCR products. The ladder information corresponding to 500 and 400 bp bands were specified as well as the primer dimer with an asterisk on each gel image. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Antibody Therapeutics.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Antibody Ther, Volume 2, Issue 2, 31 May 2019, Pages 56–63, https://doi.org/10.1093/abt/tbz007 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3 The discovery of a scFv sequence and the assembly of the CAR Figure 3 The discovery of a scFv sequence and the assembly of the CAR. (A) A simplified depiction of the acquisition ... Figure 3 The discovery of a scFv sequence and the assembly of the CAR. (A) A simplified depiction of the acquisition of the heavy and light chain’s sequences through IMGTV database and a general scFv structure. (B) A general description of a second-generation CAR design with the protein sequences of each domains. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Antibody Therapeutics.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Antibody Ther, Volume 2, Issue 2, 31 May 2019, Pages 56–63, https://doi.org/10.1093/abt/tbz007 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4 In vitro validation of CAR constructs Figure 4 In vitro validation of CAR constructs. (A) Jurkat cells were retrovirally transduced with either IGK (blue) ... Figure 4 In vitro validation of CAR constructs. (A) Jurkat cells were retrovirally transduced with either IGK (blue) or IGL CAR (red). The expression was assessed by flow cytometry detection of anti-Fab antibody. Non-transduced Jurkat cells (gray) were used as baseline control, representative of N > 2. (B) Activated primary T-cells were retrovirally transduced with either IGK (blue) or IGL CAR (red). The expression was assessed by flow cytometry, and mock transduced T cells (gray) were used as a baseline control, representative of N = 2. (C) CAR expressing T cells (IGK, blue; IGL CAR, red; and mock, black) specificity to their respective targets in functional assay. Inset: staining of the target cells with specific antibodies, MAVER-1 (Igλ+, black), BL-41 (Igκ+, light gray) and as a negative control Jurkat (Ig-, white). Cytokine secretion (TNF-α) was analyzed by flow cytometry and plotted. Data represent mean ± SD of triplicates, Student t-test where <sup>**</sup>P < 0.01, <sup>****</sup>P < 0.0001, N = 2. (D) Bioluminescence-based killing assay of CAR expressing T cells (IGK, blue; IGL CAR, red; and mock, black) against the same target cells as in (C) and plotted as percentage of lysis over time. Data represent mean ± SD of quadruplicates, Student t-test where <sup>***</sup>P < 0.001, <sup>****</sup>P < 0.0001, N = 2. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Antibody Therapeutics.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Antibody Ther, Volume 2, Issue 2, 31 May 2019, Pages 56–63, https://doi.org/10.1093/abt/tbz007 The content of this slide may be subject to copyright: please see the slide notes for details.