Antigen-binding agents that specifically bind epidermal growth factor receptor variant III

Abstract

The present disclosure relates to antigen-binding agents that specifically bind to epidermal growth factor receptor variant III (EGFRvIII). Antigen-binding agents of the present disclosure include antibodies and antigen-binding fragments thereof, chimeric antigen receptors (CARs) and bi-specific T-cell engagers (BiTE!), bispecific killer cell engagers (BiKEs) and trispecific killer cell engagers (TrikEs). Nucleic acid molecules and vectors expressing antibodies, antigen-binding fragments. CARs, BiTEs, BiKEs or TriKEs are also encompassed by the present disclosure. Immune cells engineered to express CARs, BiTEs, BiKEs or TriKEs may be used to specifically recognize and kill cells expressing EGFRvIII.

Claims

1. An antigen-binding agent comprising an antigen-binding domain of an antibody that specifically binds to epidermal growth factor receptor variant III (EGFRvIII), wherein the antigen-binding domain comprises: a. a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 15, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:16, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 17, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:19, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:20 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:21; b. a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 7, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:8, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:9, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:11, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:12 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:13, or c. a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 23, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:24, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:25, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:27, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:28 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:29.

2. The antigen-binding agent of claim 1, wherein the antigen-binding domain comprises humanized framework amino acid sequences.

3. The antigen-binding agent of claim 1, wherein the antigen-binding agent is an antibody, an antigen-binding fragment thereof, a chimeric antigen receptor, a bi-specific T-cell engager, a bispecific killer cell engager, or a trispecific killer cell engager.

4. The antigen-binding agent of claim 3, wherein the antigen-binding agent is an antibody or an antigen-binding fragment thereof which specifically binds to EGFRvIII and is selected from the group consisting of: a. an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:18 and/or a light chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 14; b. an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:10 and/or a light chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:6, or c. an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:26 and/or a light chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 22.

5. The antigen-binding agent of claim 3, wherein the antigen-binding agent is a chimeric antigen receptor, a bi-specific T-cell engager, a bispecific killer cell engager or a trispecific killer cell engager and wherein the antigen-binding domain comprises: a. an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:18 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 14; b. an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:10 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 6, or c. an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:26 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 22.

6. The antigen-binding agent of claim 5, wherein the chimeric antigen receptor further comprises: a. a transmembrane domain, wherein the antigen-binding domain is optionally connected to the transmembrane domain by a spacer; b. at least one intracellular signaling domain; and c. optionally at least one costimulatory domain.

7. The antigen-binding agent of claim 6, wherein the intracellular signaling domain is selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, and DAP12.

8. The antigen-binding agent of claim 6, wherein the chimeric antigen receptor comprises at least one costimulatory domain.

9. The antigen-binding agent of claim 8, wherein the costimulatory domain is selected from the group consisting of CD28, CD27, 4-1BB, OX40, CD7, B7-1 (CD80), B7-2 (CD86), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D and a combination thereof.

10. The antigen-binding agent of claim 5, wherein the chimeric antigen receptor comprises: a. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:33; b. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:34 or as set forth in SEQ ID NO:77; c. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:30; d. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:32 or as set forth in SEQ ID NO:74; e. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:35, or f. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:36 or as set forth in SEQ ID NO:80.

11. An isolated cell population engineered to express the chimeric antigen receptor of claim 5.

12. The isolated cell population of claim 11, wherein the isolated cell population comprises immune cells.

13. The isolated cell population of claim 12, wherein the immune cells comprise T cells, Natural Killer (NK) cells, cytotoxic T cells, regulatory T cells or a combination thereof.

14. A pharmaceutical composition comprising the isolated cell population of claim 11 and a pharmaceutically acceptable excipient.

15. A method of treating a subject having a cancer associated with EGFRvIII expression, the method comprising administering the isolated cell population of claim 11.

16. The antigen-binding agent of claim 3, wherein the antigen-binding agent is a bi-specific T-cell engager comprising; a. a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:15, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:16, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:17, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:19, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:20 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:21, and wherein the bi-specific T-cell engager optionally comprises a CDR3-specific binding domain; b. an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:18 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:14 and wherein the bi-specific T-cell engager optionally comprises a CDR3-specific binding domain or, c. wherein the bi-specific T-cell engager optionally comprises a CDR3-specific binding domain and an amino acid sequence at least 80% identical to SEQ ID NO:82.

17. The antigen-binding agent of claim 1, wherein the antigen-binding domain is in the form of a single chain variable fragment (scFv).

18. The antigen-binding agent of claim 17, wherein the scFv structure is defined by the formula VH-linker-VL.

19. An isolated nucleic acid molecule encoding the antigen-binding agent of claim 1.

20. A vector comprising a nucleic acid molecule encoding the antigen-binding agent of claim 1.

21. An isolated cell comprising or expressing the antigen-binding agent of claim 1.

22. A method of treating subject having a cancer associated with EGFRvIII expression, the method comprising administering the antigen-binding agent of claim 1.

23. The antigen-binding agent of claim 1, wherein the antigen-binding agent comprises: a. the amino acid sequence of set forth in SEQ ID NO:45; b. an amino acid sequence of at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:45 and comprising a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:15, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 16, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:17, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:19, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 20 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:21; b. the amino acid sequence of set forth in SEQ ID NO:44; c. an amino acid sequence of at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:44 and comprising a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:7, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:8, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:9, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:11, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 12 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:13; c. the amino acid sequence of set forth in SEQ ID NO:46; or d. an amino acid sequence of at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:46 and comprising CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:23, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:24, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:25, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:27, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 28 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:29.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-1E: represents histograms obtained using flow cytometry on supernatants of selected hybridomas on U87MG cell lines overexpressing wild type human EGFR (U87WT) or EGFRvIII (U87vIII) as indicated.

(2) FIG. 2: is an alignment between the amino acid sequence of the extracellular domains of wild-type human EGFR (EGFR WT, SEQ ID NO: 2) and EGFRvIII (SEQ ID NO: 91).

(3) FIGS. 3A and 3B: show the results of anti-hEGFRvIII mAbs binding properties to various fragments of the EGFRvIII and WT protein cells displayed on yeast cells (+++ represents 95%+/5% positive yeast cells which is characterized by positive antibody binding with high affinity; ++ represents 70%+/20% positive yeast cells which is characterized by positive antibody binding with medium affinity; + represents 30%+/20% positive yeast cells which is characterized by positive antibody binding with low affinity; (+) represents 5-9% positive yeast cells which is characterized by positive antibody binding with very low affinity; +/ represents less than 5% positive yeast cells which is characterized by ambiguous antibody binding, represents 0% positive yeast cells which is characterized by no binding; nt=not tested

(4) FIG. 4: Schematic illustrating the synthetic assembly and sequence of the 5B7 antigen-binding domain and mouse CD8 hinge (SEQ ID NO: 30) for insertion in CAR vector with the heavy chain portion (underlined), amino acids corresponding to a restriction site (*), linker (double underlined), the light chain portion (underlined).

(5) FIG. 5A: Schematic illustrating different elements and DNA sequences of the modular CAR vector (SEQ ID NO:37), representing an example of CAR cloning strategy.

(6) FIG. 5B: Schematic illustrating 1D2-CAR-TRIM which contains a shorter scFv portion (SEQ ID NO:78)

(7) FIG. 5C: Graph comparing 1D2-CAR and 1D2-CAR-TRIM construct using the CAR-J assay. Briefly, Jurkat cells were electroporated with plasmids expressing either full length or trimmed form of the CAR as illustrated in 5B. CAR-J cells were then mixed with EGFRvIII expressing target cells and examined for activation signature (CD69 expression) via antibody staining and flow cytometric analysis.

(8) FIG. 5D: Schematic illustrating potential bi-specific T-cell engagers and bispecific killer cell engagers.

(9) FIGS. 6A and 6B: Human CD8 T cell line Jurkat transiently transfected with the EGFRvIII CAR construct which also expressed green fluorescent protein (GFP) using a 2A self-cleaving peptide based multi-gene expressing system. Expression of GFP was quantified using flow cytometry (A) and the surface expression of CAR was detected by flow cytometry using a fluorescent tagged antibody that recognize murine IgG (Goat Anti-Mouse IgG H&L [Alexa Fluor 594]; Abcam) (B) (5B7=F265-5B7; 3D12=F269-3D12; 1D2=F271-1D2).

(10) FIG. 7: Schematic illustrating the high-throughput screening method for CAR functionality. (A) Jurkat cells electroporated with various CAR constructs were exposed to varying doses of target cells with (U87-vIII, DKMG) or without (U87-MG) specific target expression. CAR constructs found to have inappropriate properties for advancement are also shown (X1, X2) (B) Tonic signaling/auto-activation of each CAR construct was assessed by examining the level of CD69 expression in CAR-expressing (GFP+) cells without additional stimulation. (C and D) The relative increase in expression of CD69 within CAR cells following stimulation with EGFRvIII expressing target cells is shown.

(11) FIG. 8A: Graph illustrating the in vitro functionality of EGFRvIII CAR-T constructs. Jurkat cells electroporated with various CAR constructs (EGFRvIII 5B7 CAR, EGFRvIII 3D12 CAR, EGFRvIII 1D2 CAR, EGFR WT CAR) were exposed to target cells with (U87vIII) or without (U87, OVCAR3 and A20) EGFRvIII target expression for 24 hours. The level of cell activation was measured by quantifying surface expression of CD69 on Jurkat cells (CD45 positive) by flow cytometry.

(12) FIG. 8B: Graph illustrating screening of CAR functionality with EGFRvIII-specific CAR constructs containing hinge elements of varying amino acid length (screening hinge truncations in EGFRvIII-CAR). Jurkat cells electroporated with various CAR constructs which contained hinge elements ranging from very long, L17-CD8h which contains the entire human CD8 hinge domain and an extended glycine linker, to very short, 1CD8h which contains a single C-terminal amino acid from the human CD8-hinge sequence. Jurkat-cells transiently expressing these various CAR constructs were exposed to target cells with (U87-vIII) or without (U87-MG) specific target expression. Target-induced activation of each CAR construct was assessed by examining the level of CD69 expression in CAR-expressing (GFP+) cells using human CD69-specific antibody staining and flow cytometry.

(13) FIG. 9: Graphs showing the tumor volume (meanSEM) in athymic nude mice (Jackson Laboratory, Barr Harbor, ME) injected subcutaneously with 110.sup.6 (FIG. 9A) or 410.sup.6 (FIG. 9B) U87vIII human glioblastoma cells expressing EGFRvIII and administered with 10.sup.5 (FIG. 9A) or 10.sup.6 (FIG. 9B) human primary T cells expressing EGFRvIII 1D2 CAR or negative control CD19 CAR on day 5 post tumor cell injection. Tumor size (the length and the width) was measured using a digital vernier caliper. Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter. The level of CD8 T cell infiltration in tumors from two animals each from groups receiving EGFRvIII targeted CAR-T and showing good tumor control was assessed and compared to CD19 CAR-T negative control (FIG. 9C).

(14) FIG. 10: Schematic illustrating the generation of EGFRvIII targeted NK92-CAR cells for in vitro testing of tumor cytotoxicity (FIG. 10A). Graphs showing data of cytotoxicity of control NK92 cells (NK92-no CAR), NK 92 cells expressing EGFRvIII 3D12 CAR construct (NK92-3D12CAR) or EGFR WT CAR construct towards cells expressing EGFRvIII (U87-vIII) or WT EGFR (U87) as measured by LDH (FIG. 10B) or .sup.51Cr-release assay (FIG. 10C). Wild-type NK92 cells devoid of a CAR construct was used as negative control (NK92 wt).

(15) FIG. 11: Graphs illustrating tumor growth (left panels) and survival (right panels) of tumor-bearing athymic nude mice administered with NK92 cells expressing EGFRvIII 3D12 CAR construct or devoid of CAR expression (NK92-WT or NK92 wt). Athymic nude mice (Jackson Laboratory, Barr Harbor, ME) were injected subcutaneously with 110.sup.6 U87vIII human glioblastoma cells expressing EGFRvIII. On day 2 post tumor cell injection, mice were given either 510.sup.6 wild-type NK92 cells devoid of CAR or NK92 cells expressing EGFRvIII 3D12 CAR (FIG. 11A) or on day 3 post tumor induction animals were given 110.sup.7 EGFRvIII 3D12 NK-CAR or left untreated (FIG. 11B). Animals were monitored for survival and tumor growth. Tumor size (the length and the width) was measured using a digital vernier caliper. Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter.

(16) FIG. 12: Graph illustrating repression of target cell growth in CAR transduced T-cell/target cell co-cultures. Human primary peripheral blood derived T cells were transduced with EGFRvIII-specific CAR lentivirus (3D12-CAR-T) or treated similarly in the absence of lentivirus (Mock) and grown for several days in culture. CAR-transduced or non-transduced T cells were then placed in co-culture with EGFRvIII antigen expressing target cells which express nuclear localized mKate2-fluorescent protein (U87vIII). Cells were then examined using live fluorescence microscopy (Incucyte, Sartorius). Graph depicts the relative target cell growth over 6 days as measured via automated counting of mKate2+ cells.

(17) FIG. 13: Graphs illustrating tumor growth (left panels) and survival (right panels) of tumor-bearing NOD/SCID/IL-2Ry-null (NSG) mice. NSG mice (Jackson Laboratory, Barr Harbor, ME) were injected subcutaneously with 110.sup.6 U87vIII human glioblastoma cells expressing EGFRvIII. On day 7 post tumor cell injection, mice were given either primary human T cells transduced with EGFRvIII-3D12 CAR or left untreated. Tumour growth was then monitored by caliper measurements (FIG. 13A). Tumor size (the length and the width) was measured using a digital vernier caliper. Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter. Survival is defined as time to humane endpoint (defined as a tumour volume exceeding 2000 mm.sup.3) with and without CAR-T treatment (FIG. 13B).

(18) FIG. 14: Graph illustrating screening of bi-specific T cell engager activity with EGFRvIII-specific constructs containing 3D12-scFV sequence linked to OKT3 human CD3-specific scFV. Supernatant from human embryonic kidney (HEK293) cells transiently expressing 3D12-OKT3 bi-specific engager construct were transferred to wells containing Jurkat cells and varying doses of antigen expressing target cells (U87vIII). Target-induced activation of T cells in the presence or absence of bispecific T-cell engager was measured by examining the level of CD69 expression using human CD69-specific antibody staining and flow cytometry. The fold change in CD69 expression with and without bispecific-T cell engager over varying doses of target cell is shown here.

(19) FIG. 15: Graph illustrating repression of target cell growth in T-cell/target cell co-cultures in the presence of a bi-specific T cell engager constructs containing 4E11-scFV sequence linked to OKT3 human CD3-specific scFV. A control bi-specific T cell engager composed of humanCD19-specific scFv linked to OKT3 is shown for comparison. Supernatants from human embryonic kidney cells transiently expressing 4E11-OKT3 bi-specific T cell engager construct or the CD19-OKT3 bi-specific T cell engager construct were transferred to wells containing primary blood derived T cells and EGFRvIII antigen expressing target cells which express nuclear localized mKate2-fluorescent protein (U87vIII). Cells were then examined using live fluorescence microscopy (Incucyte, Sartorius, USA). Graph depicts the relative target cell growth over 72 hours as measured via automated counting of mKate2+ cells.

DETAILED DESCRIPTION

(20) As used herein the term EGFRvIII or vIII refers to epidermal growth factor receptor variant III.

(21) As used herein the term EGFR refers to human epidermal growth factor receptor. The term wt EGFR, WT EGFR, EGFR WT or EGFR wt are used interchangeably and refers to wild-type EGFR.

(22) The use of the terms a and an and the and similar referents in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

(23) Unless specifically stated or obvious from context, as used herein the term or is understood to be inclusive and covers both or and and.

(24) The term and/or where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other.

(25) The terms comprising, having, including, and containing are to be construed as open-ended terms (i.e., meaning including, but not limited to) unless otherwise noted. The term consisting of is to be construed as close-ended. The term consisting essentially of when used in the context of CDR sequences means that the CDR sequence may be slightly (e.g., +/1 or 2 aa) longer or shorter.

(26) As used herein the term native with respect to a protein such as EGFRvIII or EGFR refers to the natural conformation of the protein and includes proteins that are properly folded and/or functional.

(27) As used herein the term denatured with respect to a protein such EGFRvIII or EGFR refers to a protein that has lost its natural conformation and may entail for example, a loss in the tertiary and secondary structure.

(28) As used herein, the term antibody encompasses monoclonal antibody, polyclonal antibody, humanized antibody, chimeric antibody, human antibody, domain antibody, multispecific antibody (e.g., bispecific antibodies such as for example a bi-specific T-cell engager) etc. The term antibody encompasses molecules that have a format similar to those occurring in nature (e.g., human IgGs, etc.).

(29) As used herein the term not able to significantly bind a peptide means that the binding is between 0% and 15% of that observed for the non-mutated EGFRvIII peptide (SEQ ID NO:5).

(30) As used herein the term antigen-binding domain refers to the domain of an antibody or of an antigen-binding fragment which allows specific binding to an antigen.

(31) The antigen-binding domain of the present disclosure may comprise for example, at least one complementarity determining region of the antibody heavy chain. In accordance with the present disclosure, the antigen-binding domain may comprise at least two complementarity determining regions of the antibody heavy chain. Further in accordance with the present disclosure, the antigen-binding domain may comprise three complementarity determining regions of antibody heavy chain. The antigen-binding domain may comprise or also comprise at least one complementarity determining region of the antibody light chain. In accordance with the present disclosure, the antigen-binding domain may comprise at least two complementarity determining regions of the antibody light chain. Further in accordance with the present disclosure, the antigen-binding domain may comprise at least three complementarity determining regions of the antibody light chain. The antigen-binding domain may comprise three complementarity determining regions of the light chain and three complementarity determining regions of the heavy chain of the antibody.

(32) An exemplary embodiment of a molecule comprising an antigen-binding domain is an antibody or an antigen-binding fragment thereof.

(33) Another exemplary embodiment of a molecule comprising an antigen-binding domain is a chimeric antigen receptor.

(34) Yet another exemplary embodiment of a molecule comprising an antigen-binding domain is a bi-specific T-cell engager.

(35) Yet a further exemplary embodiment of a molecule comprising an antigen-binding domain is a bispecific killer cell engager.

(36) Another exemplary embodiment of a molecule comprising an antigen-binding domain is a trispecific killer cell engager.

(37) A further exemplary embodiment of a molecule comprising an antigen-binding domain is an antigen-binding fragment such as for example a single chain Fv. In accordance with the present disclosure, the single chain Fv comprises for example, the heavy chain variable region and the light chain variable region of an antibody that specifically binds to EGFRvIII. The heavy chain variable region and the light chain variable region of the antibody may be connected by a linker.

(38) Linker sequences include for example, an amino acid sequence of at least 5 amino acids. Multimers of the pentapeptide G.sub.4S (SEQ ID NO: 88) (e.g., (Gly.sub.4Ser), where n is a positive integer of 1 or more) are often used in the engineering of scFvs. Those include for example, a 15-mer (G.sub.4S).sub.3 (SEQ ID NO: 89) found in some of the first scFv fragments (Huston et al., 1988), an 18-mer GGSSRSSSSGGGGSGGGG (SEQ ID NO: 87) (Andris-Widhopf et al., 2011) and the 20-mer (G.sub.4S).sub.4 (SEQ ID NO: 90) (Schaefer et al., 2010). Many other sequences have been proposed, including sequences with added functionalities, e.g. an epitope tag or an encoding sequence containing a Cre-Lox recombination site (Sblattero & Bradbury, 2000) or sequences improving scFv properties, often in the context of particular antibody sequences. The linker of the present disclosure may have the sequence GGGSGGGGSGGGGS (SEQ ID NO:47).

(39) Chimeric Antigen Receptors

(40) The present disclosure relates to chimeric antigen receptors that specifically bind to EGFRvIII and nucleic acid encoding same. The chimeric antigen receptors of the present disclosure comprise an antigen-binding domain of an antibody that specifically binds to EGFRvIII.

(41) The basic structure of chimeric antigen receptors has been described in the literature (e.g., Gacerez, A. T. et al., J Cell Physiol. 231(12): 2590-2598 (2016), Sadelain, M. et al. Cancer Discovery, 3(4): 388-98, (2013), Zhang, C. et al., Biomarker Research, 5:22 (2017)).

(42) Chimeric antigen receptors have an extracellular region (or ectodomain) which comprises an antigen-binding domain and an intracellular region (or endodomain) which comprises the transmembrane domain and the intracytoplasmic domain which comprise intracellular signaling domains of immune response pathways or immune effector function (e.g., cytolytic activity, helper activity including secretion of cytokines).

(43) The general structure of the chimeric antigen receptors of the present disclosure is composed of an antigen-binding domain, a transmembrane domain, an optional costimulatory domain and an intracellular signaling domain.

(44) The antigen-binding domain is generally composed of an antibody's heavy chain variable region and light chain variable region connected via a linker and forming, for example, a single chain (e.g., scFv). The antibody used to generate the CAR construct is selected for its ability to specifically bind to epidermal growth factor receptor variant III (EGFRvIII) while not binding to wild-type EGFR.

(45) The heavy chain variable region may be at the N-terminus of the polypeptide chain, followed by the linker and the light chain variable region. Alternatively, the light chain variable region may be at the N-terminus of the polypeptide chain, followed by the linker and the heavy chain variable region. In some instances, the single chain Fv may also comprises portions of the constant region.

(46) Chimeric antigen receptors may also comprise a hinge region or spacer which connects the antigen-binding domain and the transmembrane domain. The spacer may allow a better presentation of the antigen-binding domain at the surface of the cell.

(47) In accordance with the present disclosure, the spacer may be optional. Alternatively, the spacer may comprise for example, between 1 to 200 amino acid residues, typically between 10 to 100 amino acid residues and more typically between 25 to 50 amino acid residues. The spacer may originate from a human protein.

(48) In accordance with the present disclosure, the spacer or hinge region may be, for example and without limitation a CD8 hinge (e.g., mouse, human CD8) or an IgG hinge (a human immunoglobulin hinge) or combination thereof.

(49) An exemplary embodiment of a linker and hinge combination is provided in SEQ ID NO:81 where the hinge portion is from human CD8.

(50) The transmembrane domain allows the extracellular region of the CAR to be anchored at the cell membrane. The transmembrane domain may be natural or synthetic and usually comprises hydrophobic amino acid residues. The transmembrane domain may be obtained from any naturally occurring protein having a transmembrane domain. The transmembrane domain is particularly selected for its ability to signal to the intracellular domain that the antigen-binding domain has bound to its target.

(51) Exemplary embodiments of transmembrane domains include, for example and without limitation, the alpha, beta or CD3zeta chain of the T-cell receptor complex, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

(52) In some embodiments, the transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137). GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1). NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB 1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226). SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile). CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55). PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C.

(53) A particular embodiment of transmembrane domain is the transmembrane domain of CD28.

(54) In accordance with the present disclosure, the chimeric antigen receptors may comprise one or more intracellular signaling domain derived from CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, or DAP12.

(55) Chimeric antigen receptors may also optionally comprise at least one costimulatory domain.

(56) In accordance with the present disclosure, the costimulatory domain may be, for example, from CD28, CD27, 4-1BB, OX40, CD7, B7-1 (CD80), B7-2 (CD86), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D or a combination thereof.

(57) In order to be targeted to the secretory pathway, the chimeric antigen receptor may also comprise a signal peptide such as, for example, a signal peptide of CD28 or any other signal peptide suitable for immune cells. The signal peptide is cleaved (cleavable).

(58) The antigen-binding fragments of the present disclosure may comprise human or humanized framework amino acid sequences.

(59) Antibodies or Antigen-Binding Fragments

(60) Typically, an antibody is constituted from the pairing of two light chains and two heavy chains. Different antibody isotypes exist, including IgA, IgD, IgE, IgG and IgM. Human IgGs are further divided into four distinct sub-groups namely; IgG1, IgG2, IgG3 and IgG4. Therapeutic antibodies are generally developed as IgG1 or IgG2.

(61) In an exemplary embodiment, the antibody or antigen-binding fragment of the present disclosure may comprise, for example, a human IgG1 constant region. In another exemplary embodiment, the antibody or antigen-binding fragment of the present disclosure may comprise, for example, a human IgG2 constant region.

(62) The light chain and heavy chain of human antibody IgG isotypes each comprise a variable region having 3 hypervariable regions named complementarity determining regions (CDRs). The light chain CDRs are identified herein as CDRL1 or L1, CDRL2 or L2 and CDRL3 or L3. The heavy chain CDRs are identified herein as CDRH1 or H1, CDRH2 or H2 and CDRH3 or H3. Complementarity determining regions are flanked by framework regions (FR) in the order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. CDRs may be identified using for example, the Kabat and Chotia definitions (Kabat, J. Immunol. 1991, Chotia and Lesk 1987). However, others (Abhinandan and Martin, 2008) have used modified approaches based loosely on Kabat and Chotia resulting in the delineation of shorter CDRs. Lefranc also discloses the IMGT numbering scheme for CDRs (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999)).

(63) The overall binding affinity of the antibody or antigen-binding fragment thereof is often dictated by the sequence of the CDRs. The framework regions may also play a role in the proper positioning and alignment in three dimensions of the CDRs for optimal antigen-binding.

(64) As used herein an antigen-binding fragment refers to a fragment of an antibody that may be obtained by enzymatic digestion of an antibody, by recombinant DNA technology and the like. Antigen-binding fragments thereof of the present disclosure encompass molecules having an antigen-binding domain comprising amino acid residues that confer specific binding to an antigen (e.g., one or more CDRs).

(65) Examples of antigen-binding fragments encompassed by the present disclosure include (i) a Fab fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; (ii) a F(ab).sub.2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment consisting of the V.sub.L and V.sub.H domains of a single arm of an antibody (e.g. scFv), (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V.sub.H domain; and (vi) an isolated complementarity determining region (CDR), e.g., V.sub.H CDR3.

(66) Although the two domains of the Fv fragment, V.sub.L and V.sub.H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single polypeptide chain in which the V.sub.L and V.sub.H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

(67) Single chain antibodies (e.g., single domain), diabody, minibody, nanobody and the like are encompassed within the term antigen-binding fragment. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for activity in the same manner as for intact antibodies.

(68) Particular embodiments of antigen-binding fragments may include for example, a scFv, a Fab, a Fab or a (Fab).sub.2.

(69) The term humanized antibody encompasses fully humanized (i.e., frameworks are 100% humanized) and partially humanized sequences (e.g., at least one variable region contains one or more amino acids from a human antibody, while other amino acids are amino acids of a non-human parent antibody). Typically, a humanized antibody or antigen-binding fragment contains CDRs of a non-human parent antibody (e.g., mouse, rat, rabbit, non-human primate, etc.) and frameworks that are identical to those of a natural human antibody or of a human antibody consensus. In such instance, those humanized antibodies or antigen-binding fragments are characterized as fully humanized. A humanized antibody or antigen-binding fragment may also contain one or more amino acid substitutions that have no correspondence to those of the human antibody or human antibody consensus. Such substitutions include, for example, back-mutations (e.g., re-introduction of non-human amino acids) that may preserve the antibody characteristics (e.g., affinity, specificity etc.). Such substitutions are usually in the framework region. A humanized antibody or antigen-binding fragment usually also comprise a constant region (Fc) or a portion thereof which is typically that of a human antibody. Typically, the constant region of a humanized antibody or antigen-binding fragment is identical to that of a human antibody. A humanized antibody may be obtained by CDR grafting (Tsurushita et al, 2005; Jones et al, 1986; Tempest et al, 1991; Riechmann et al, 1988; Queen et al, 1989). Such antibody is considered as fully humanized.

(70) The term chimeric antibody refers to an antibody having a constant region from an origin distinct from that of the parent antibody. The term chimeric antibody encompasses antibodies having a human constant region. Typically, a chimeric antibody is composed of variable regions originating from a mouse antibody and of human constant regions.

(71) The term hybrid antibody refers to an antibody comprising one of its heavy or light chain variable region (its heavy or light chain) from a certain type of antibody (e.g., humanized) while the other of the heavy or light chain variable region (the heavy or light chain) is from another type (e.g., murine, chimeric).

(72) Antibodies and/or antigen-binding fragments of the present disclosure may originate, for example, from a mouse, a rat or any other mammal or from other sources such as through recombinant DNA technologies. Antibodies or antigen-binding fragment of the present disclosure may include for example, a synthetic antibody, a non-naturally occurring antibody, an antibody obtained following immunization of a non-human mammal etc.

(73) Antibodies or antigen-binding fragments thereof of the present disclosure may be isolated and/or substantially purified.

(74) Variants

(75) The present disclosure also encompasses variants of the antigen-binding agents described herein. Variant of the present disclosure include those having a variation in their amino acid sequence, e.g., in one or more CDRs, in one or more framework regions and/or in the constant region. Variant included in the present disclosure are those having, for example, similar or improved binding affinity in comparison with an original antigen-binding agent.

(76) Exemplary variants encompassed by the present disclosure are those which may comprise an insertion, a deletion or an amino acid substitution (conservative or non-conservative). These variants may have at least one amino acid residue in its amino acid sequence removed and a different residue inserted in its place.

(77) More particularly, variants encompassed by the present disclosure include those having a light chain variable region and/or a heavy chain variable region having at least 80% sequence identity with the light chain variable region and/or a heavy chain variable region of the antibodies or antigen-binding fragments, CARs, BiTEs, BiKEs or TriKEs disclosed herein. The CDRs of the variants of the present disclosure may be identical to those of the antibodies or antigen-binding fragments, CARs, BiTEs, BiKEs or TriKEs disclosed herein.

(78) Also encompassed by the present disclosure are variants having CDRs amino acid residues that are identical and framework regions that are at least 80% sequence identical to those of the antigen-binding domain, antibody or antigen-binding fragment disclosed herein.

(79) Conservative substitutions may be made by exchanging an amino acid residue (of a CDR, variable chain, framework region or constant region, etc.) from one of the groups listed below (group 1 to 6) for another amino acid of the same group.

(80) Other exemplary embodiments of conservative substitutions are shown in Table A. (group 1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile) (group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr) (group 3) acidic: Aspartic acid (Asp), Glutamic acid (Glu) (group 4) basic: Asparagine (Asn), Glutamine (Gln), Histidine (His), Lysine (Lys), Arginine (Arg) (group 5) residues that influence chain orientation: Glycine (Gly), Proline (Pro); and (group 6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)

(81) Non-conservative substitutions will entail exchanging a member of one of these groups for another.

(82) TABLE-US-00001 TABLE A Amino acid substitution Original Exemplary Conservative residue substitution substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg, Asp Gln Asp (D) Glu, Asn Glu Cys (C) Ser, Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp, Gln Asp Gly (G) Ala Ala His (H) Asn, Gln, Lys, Arg, Arg Ile (I) Leu, Val, Met, Ala, Leu Phe, norleucine Leu (L) Norleucine, Ile, Val, Ile Met, Ala, Phe Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Leu norleucine

(83) Percent identity is indicative of amino acids which are identical in comparison with the original peptide and which may occupy the same or similar position. Percent similarity will be indicative of amino acids which are identical and those which are replaced with conservative amino acid substitution in comparison with the original peptide at the same or similar position.

(84) Generally, the degree of similarity and identity between variable chains has been determined herein using the Blast2 sequence program (Tatiana A. Tatusova, Thomas L. Madden (1999), Blast 2 sequencesa new tool for comparing protein and nucleotide sequences, FEMS Microbiol Lett. 174:247-250) using default settings, i.e., blastp program, BLOSUM62 matrix (open gap 11 and extension gap penalty 1; gapx dropoff 50, expect 10.0, word size 3) and activated filters.

(85) Variants of the present disclosure therefore comprise those which may have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an original sequence or a portion of an original sequence.

(86) Nucleic Acids, Vectors and Cells

(87) As used herein, the term nucleic acid refers to RNA, DNA, cDNA and the like.

(88) The present disclosure encompasses nucleic acids capable of encoding any of the CDRs, light chain variable regions, heavy chain variable regions, light chains, heavy chains, scFvs antibodies or antigen-binding fragments, CARs, BiTEs, BiKEs or TriKEs or variants described herein.

(89) Nucleic acid molecules encoding CARs, BiTEs, BiKEs or TriKEs are introduced into immune cells where they are expressed. Nucleic acids encoding CARs, BiTEs, BiKEs or TriKEs may be delivered by transduction systems such as for example, using viral systems (from lentiviruses, adenoviruses, adeno-associated viruses, etc.) or non-viral systems (e.g., transposon). Exemplary system involving transposons includes the Sleeping Beauty transposon system and the piggyBac transposon system. Gene editing system may also be used including, without limitation, the CRISPR/cas system, the Zinc finger nuclease system, the Transcription Activator-Like Effector Nucleases (TALENs) system. Nucleic acids (e.g., RNA or DNA) may be delivered by other means such as by liposomes, naked DNA, polymers, electroporation etc.

(90) Due to the inherent degeneracy of the genetic code, other nucleic acid sequences that encode the same amino acid sequence may be produced and used to express the antibody or antigen-binding fragments thereof of the present disclosure. The nucleotide sequences may be engineered using methods generally known in the art in order to alter the nucleotide sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

(91) In yet another aspect, the present disclosure relates to a vector or vectors comprising the nucleic acids described herein.

(92) In accordance with the present disclosure, the vector or vectors may be an expression vector(s).

(93) Further in accordance with the present disclosure, the vector may be a viral vector. Exemplary embodiments of viral vectors include lentiviral vectors, adenoviral vectors or adeno-associated viral vectors.

(94) The expression vector usually contains the elements for transcriptional and translational control of the inserted coding sequence in a particular host. These elements may include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5 and 3 un-translated regions. Exemplary embodiments of promoter used to drive CAR construct expression in T cells includes the EF1a promoter. Other exemplary embodiments of promoters include constitutively active viral promoters such as for example, the immediate early cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR), Rous sarcoma virus (RSV) promoter.

(95) Methods that are well known to those skilled in the art may be used to construct such expression vectors. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

(96) In order to make the antibodies or antigen-binding fragments of the present disclosure, a vector or a set of vectors expressing the light chain and heavy chain are introduced into a cell.

(97) The present disclosure encompasses vectors or a set of vectors where the light chain variable region and the heavy chain variable region of the antibody or antigen-binding fragment thereof are encoded by the same nucleic acid molecule (e.g., same vector) or by separate nucleic acid molecules (e.g., separate vectors).

(98) In another aspect the present disclosure relates to an isolated cell which may comprise the nucleic acids, vectors, antibodies or antigen-binding fragment, CARs, BiTEs, BiKEs or TriKEs described herein.

(99) Yet another aspect of the present disclosure relates to an isolated host cell which expresses the antibody or antigen-binding fragment, CAR, BiTE, BiKE or TriKE of the present disclosure. The isolated host cell for CAR, BiTE, BiKEs or TriKE expression may be an immune cell such as for example and without limitation, T cells, Natural Killer (NK) cells, cytotoxic T cells, or regulatory T cells.

(100) The present disclosure also relates to a cell population engineered to express the CAR, BiTE, BiKE or TriKE of the present disclosure. The cell population may be homogenous or heterogenous. In accordance with the present disclosure, the cell population may comprise T cells (CD4+ T-cells, CD8+ T-cells or a combination thereof), Natural Killer (NK) cells, cytotoxic T cells, regulatory T cells, and combinations thereof. In accordance with the present disclosure the cell population may be autologous. In accordance with the present disclosure the cell population may be allogenic.

(101) As used herein the term autologous refers to material derived from the same individual.

(102) The term allogeneic refers to material derived from a different subject of the same species but that is genetically distinct.

(103) As used herein, the term heterogenous with reference to a cell population means that the cell population either express different chimeric antigen receptors or that the cell population comprises different types of cells.

(104) As used herein, the term homogenous with reference to a cell population means that the cell population either express the same chimeric antigen receptors or that the cell population comprises the same type of cells.

(105) The present disclosure also encompasses complement of the nucleic acids disclosed herein. It is to be understood herein that nucleic acid molecules comprising at least a portion complementary to the nucleic acid sequence disclosed herein are also encompassed by the present disclosure. Such complementary nucleic acid molecules may be used, for example, for gene amplification or detection of the nucleic acid molecule of the present disclosure and include probes or primers.

(106) Production of Cells Expressing Chimeric Antigen Receptors

(107) Methods of manufacturing or of producing immune cells expressing chimeric antigen receptors, bi-specific T-cell engagers, bispecific killer cell engagers or trispecific killer cell engagers.

(108) Immune cells of human origin may particularly be used to generate the CAR-expressing cell population of the present disclosure. The immune cells may comprise T-cells (e.g., CD4.sup.+ or CD8.sup.+), Natural Killer cells, cytotoxic T-cells, regulatory T-cells, and combinations thereof. Particularly contemplated are T-cells or NK cells.

(109) Immune cells are first isolated from a subject by various methods known to a person skilled in the art. For example, peripheral blood mononuclear cells (PBMCs) may be isolated from a subject by leukaphoresis. T-cells may be enriched and washed to separate them from the leukocytes. The different T cell subsets may be separated using beads conjugated with specific antibody or markers. CAR-T cells are often generated from the CD3.sup.+ population. T-cells are activated by various methods including for example, by antigen-presenting cells, with specific antibodies and the like (methods reviewed in Wang, X et al., Molecular TherapyOncolytics, 3:16015, 2016).

(110) The method may comprise introducing the isolated nucleic acid molecule or the vector disclosed herein into the immune cells such that the nucleic acid integrates into the genome of the cell. The cells may be transduced using viral vectors or by other means and expanded. For example, lentiviral vectors are used to transduce immune cells with CAR-expression by exposing cells directly to viral vector containing supernatant media overnight. Cells are then assessed for transduction using a fluorescent marker included in the CAR-expressing lentiviral backbone.

(111) Following purification and quality control steps, CAR-expressing immune cells are provided to a subject in need. The subject in need may be, for example, the initial donor of the immune cells.

(112) Production of the Antibodies or Antigen-Binding Fragments in Cells

(113) The antibodies that are disclosed herein can be made by a variety of methods familiar to those skilled in the art including hybridoma methodology or recombinant DNA methods.

(114) Conventional hybridoma technology entails immunizing a rodent with an antigen, isolating and fusing spleen cells with myeloma cells lacking HGPRT expression and selecting hybrid cells by hypoxanthine, aminopterin and thymine (HAT) containing media. Hybridoma are screened to identify those producing antibodies that are specific for a given antigen. The hybridoma is expanded and cloned. The nucleic acid sequence of the light chain and heavy chain variable regions is obtained by standard sequencing methodology and expression vectors comprising the light chain and heavy chain nucleic acid sequence of an antibody are generated.

(115) For recombinant expression of antibodies, host cells are transformed with a vector or a set of vectors comprising the nucleic acid sequence of the light chain and heavy chain of the antibody or antigen-binding fragment thereof (on the same vector or separate vectors).

(116) For long-term production of recombinant proteins in mammalian systems, stable expression in cell lines may be effected. For example, nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may be transformed into cell lines using expression vectors that may contain viral origins of replication and/or endogenous expression elements and a selectable or visible marker gene on the same or on a separate vector. The disclosure is not to be limited by the vector or host cell employed. In certain embodiments of the present disclosure, the nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may each be ligated into a separate expression vector and each chain expressed separately. In another embodiment, both the light and heavy chains able to encode any one of a light and heavy immunoglobulin chains described herein may be ligated into a single expression vector and expressed simultaneously.

(117) Immunological methods for detecting and measuring the expression of polypeptides are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), fluorescence activated cell sorting (FACS) or flow cytometry. Those of skill in the art may readily adapt these methodologies to the present disclosure.

(118) Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., Chinese Hamster Ovary (CHO), HeLa, MDCK, HEK293, and W138) are available commercially and from the American Type Culture Collection (ATCC) and may be chosen to ensure the correct modification and processing of the expressed polypeptide.

(119) Typically, antibody or antigen-binding fragments thereof are produced in CHO cells, NS0 murine myeloma cells, PER.C6 human cells.

(120) The present disclosure relates to a method of making an antibody or an antigen-binding fragment thereof comprising expressing the light chain and heavy chain of the antibody or antigen-binding fragment of the present disclosure in cultured cells.

(121) The method may further comprise purifying or isolating the antibody or antigen-binding fragment of the present disclosure. The method may also further comprise conjugating the antibody or antigen-binding fragment of the present disclosure to a cargo molecule such as a therapeutic or detectable moiety.

(122) Antibody Conjugates

(123) The antibody or antigen-binding fragment thereof of the present disclosure may be linked to a cargo molecule. Exemplary embodiments of cargo molecules include without limitation a therapeutic moiety a detectable moiety, a polypeptide (e.g., peptide, enzyme, growth factor), a polynucleotide, liposome, nanoparticle, nanowire, nanotube, quantum dot, etc.

(124) More particularly, the antibody or antigen-binding fragment thereof of the present disclosure may be conjugated with a therapeutic moiety. The therapeutic moiety is usually attached to the antibody via a linker which may be cleavable or non-cleavable.

(125) Included amongst the list of therapeutic moiety are cytotoxic agents, cytostatic agents, anti-cancer agents (chemotherapeutics) and radiotherapeutics (e.g. radioisotopes).

(126) Exemplary embodiments of cytotoxic agents include, without limitation, alpha-amanitine, cryptophycin, duocarmazine, duocarmycin, chalicheamicin, deruxtecan, pyrrolobenzodiazepine (PBD), dolastatins, pseudomonas endotoxin, ricin, auristatins (e.g., monomethyl auristatin E, monomethyl auristatin F), maytansinoids (e.g., mertansine) and analogues.

(127) Exemplary embodiments of radiotherapeutics include without limitation, Yttrium-90, Scandium-47, Rhenium-186, Iodine-131, Iodine-125, and many others recognized by those skilled in the art (e.g., lutetium (e.g., Lu.sup.177), bismuth (e.g., Bi.sup.213), copper (e.g., Cu.sup.67)), astatine-211 (211At) actinium 225 (Ac.sup.225).

(128) Exemplary embodiments of chemotherapeutics include, without limitation, 5-fluorouracil, adriamycin, irinotecan, taxanes, carboplatin, cisplatin, etc.

(129) The antibody or antigen-binding fragment of the present disclosure may also be conjugated with a detectable moiety (i.e., for detection or diagnostic purposes).

(130) A detectable moiety comprises agents detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical and/or other physical means. A detectable moiety may be coupled either directly and/or indirectly (for example via a linkage, such as, without limitation, a DOTA or NHS linkage) to antibodies and antigen-binding fragments thereof of the present disclosure using methods well known in the art. A wide variety of detectable moieties may be used, with the choice depending on the sensitivity required, ease of conjugation, stability requirements and available instrumentation. A suitable detectable moiety include, but is not limited to, a fluorescent label, a radioactive label (for example, without limitation, .sup.125I, In.sup.111, Tc.sup.99, I.sup.131 and including positron emitting isotopes for PET scanner etc), a nuclear magnetic resonance active label, a luminescent label, a chemiluminescent label, a chromophore label, an enzyme label (for example and without limitation horseradish peroxidase, alkaline phosphatase, etc.), quantum dots and/or a nanoparticle. Detectable moiety may cause and/or produce a detectable signal thereby allowing for a signal from the detectable moiety to be detected.

(131) Pharmaceutical Compositions

(132) The present disclosure relates to a pharmaceutical composition which may comprise the antibodies or antigen binding fragments thereof, BiTEs, BiKEs or TriKEs of the present disclosure. The pharmaceutical composition may comprise, for example, diluents and/or other components such as immunomodulatory antibodies including but not limited to immune checkpoint blocking antibodies, cytokines or chemokines.

(133) The present disclosure relates to a pharmaceutical composition which may comprise a cell population engineered to express CARs, BiTEs, BiKEs or TriKEs of the present disclosure. The pharmaceutical composition may comprise, for example, diluents and/or other components such as immunomodulatory antibodies including but not limited to immune checkpoint blocking antibodies, cytokines or chemokines.

(134) The present disclosure also relates to pharmaceutical compositions comprising the antibodies or antigen-binding fragments (conjugated or not) disclosed herein.

(135) In addition to the active ingredients, a pharmaceutical composition may contain pharmaceutically acceptable carriers comprising without limitation, water, PBS, salt solutions, gelatins, oils, alcohols, and other excipients and auxiliaries that facilitate processing of the active compounds into preparations that may be used pharmaceutically. In instances where the pharmaceutical composition comprises live cells (e.g., human NK cell lines) the preparation may be irradiated. Pharmaceutical compositions may also contain, without limitation; dextran, dextrose, dimethylsulfoxide (DMSO), human serum albumin, PlasmaLyte A, sodium chloride

(136) As used herein, pharmaceutical composition means therapeutically effective amounts of the agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. A therapeutically effective amount as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts). Solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens, dimethylsulfoxide), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the disclosure are particulate compositions coated with polymers (e.g., poloxamers or poloxamines).

(137) Other embodiments of the compositions of the disclosure incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intracranially and intratumorally.

(138) Further, as used herein pharmaceutically acceptable carrier or pharmaceutical carrier are known in the art and include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

(139) For any compound, the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans. These techniques are well known to one skilled in the art and a therapeutically effective dose refers to that amount of active ingredient that ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating and contrasting the ED.sub.50 (the dose therapeutically effective in 50% of the population) and LD.sub.50 (the dose lethal to 50% of the population) statistics. Any of the therapeutic compositions described above may be applied to any subject in need of such therapy, including, but not limited to, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and humans.

(140) Pharmaceutical compositions comprising cells may be administered by infusion (e.g., by intravenous route, intracerebral injection or other routes). Pharmaceutical compositions comprising antibodies of the present disclosure may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

(141) In certain instances, the CAR cell population may be administered concurrently in combination with other treatments given for the same condition including for example anti-cancer agents. Exemplary embodiments of anti-cancer agents include for example and without limitation, therapeutic antibodies, immunomodulators (immune checkpoint blocking antibodies), anti-mitotics (e.g., taxanes), platinum-based agents (e.g., cisplatin), DNA damaging agents (eg. Doxorubicin) and other anti-cancer therapies that are known to those skilled in the art.

(142) Additional aspects of the disclosure relate to kits which may include vial(s) containing one or more nucleic acid encoding the CARs, BiTEs, BiKEs, TriKEs or antibodies or antigen-binding fragments described herein.

(143) Methods of Use

(144) Aspects of the disclosure comprise administering antibodies or antigen binding fragments thereof, CAR, BiTE, BiKE or TriKE molecules to a subject in need.

(145) Other aspects of the disclosure comprise administering immune cells engineered to express the CAR, BiTE, BiKE or TriKE molecules to a subject in need.

(146) The CAR, BiTE, BiKE or TriKE constructs of the present disclosure may be used to re-target engineered immune cells towards EGFRvIII-positive tumors.

(147) The engineered immune cells may be administered to a subject in need.

(148) In accordance with an aspect of the present disclosure, immune cells are isolated from the subject, engineered to express the CAR, BiTE, BiKE or TriKE construct and re-administered to the same subject.

(149) As used herein the term subject encompasses humans and animals such as non-human primates, cattle, rabbits, mice, rats, sheep, goats, horses, birds, etc. The term subject particularly encompasses humans.

(150) Subjects in need which would benefit from treatment include humans having tumor cells expressing EGFRvIII. More particularly, the immune cells engineered to express the CAR, BiTE, BiKE or TriKE construct disclosed herein may be administered to a subject suspected of having glioblastoma multiforme (GBM). Subjects in need also encompass those having or suspected of having carcinomas, such as breast carcinoma, ovarian carcinoma, prostate carcinoma, or non-small cell lung carcinomas.

(151) The term treatment for purposes of this disclosure refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. Particularly, subjects in need include subjects with an elevated level of one or more cancer markers.

(152) The present disclosure more particularly relates to a method of treating a subject having or suspected of having cancer by administering a cell population expressing the chimeric antigen receptor or the antibody or antigen-binding fragment thereof disclosed herein.

(153) The cell population expressing the chimeric antigen receptor, or the antibody or antigen-binding fragment thereof may be administered as a pharmaceutical composition either alone or in combination with other anti-cancer drugs.

(154) Other aspects of the disclosure relate to a method for detecting EGFRvIII, the method may comprise contacting a cell expressing EGFRvIII, or a sample (biopsy, a body fluid such as serum, plasma, urine etc.) comprising or suspected of comprising EGFRvIII with the antibody or antigen-binding fragments described herein and measuring binding. The sample may originate from a mammal (e.g., a human) which may have cancer (e.g., glioblastoma multiforme or carcinoma) or may be suspected of having such cancer. The sample may be a tissue sample obtained from the mammal or a cell culture supernatant.

(155) In accordance with the disclosure the sample may be a serum sample, a plasma sample, a blood sample or ascitic fluid obtained from the mammal.

(156) Further scope, applicability and advantages of the present disclosure will become apparent from the non-restrictive detailed description given hereinafter. It should be understood, however, that this detailed description, while indicating exemplary embodiments of the disclosure, is given by way of example only, with reference to the accompanying drawings.

EXAMPLES

Example 1: Generation of EGFRvIII Specific Monoclonal Antibodies

(157) Monoclonal antibodies (mAb) against EGFRvIII were generated by immunizing mice with the extracellular domain of recombinant proteins.

(158) Immunizations

(159) Mice were bled (pre-immune serum) and injected intraperitoneally and subcutaneously with 100 g of recombinant EGFRvIII protein emulsified in Titermax adjuvant (Cedarlane Labs, Burlington, ON) at day 0 and in PBS without adjuvant at day 22. Blood was collected in microvette CB 300Z (Sarstedt, Montreal, QC) at day 29, and serum was stored at 20 C. until further use. ELISA (serum titer determination)

(160) Pre- and post-immune sera titers of animals were assessed by ELISA on recombinant

(161) EGFRvIII protein. Unless otherwise stated, all incubations were performed at room temperature. Briefly, half-area 96-well plates (Costar #3690) were coated with 25 l per well of immunogen at 5 g/ml in PBS and incubated overnight at 4 C. Microplates were washed three times in PBS and blocked for 30 min with PBS containing 1% bovine serum albumin (BSA, Sigma Cat #A7030). Blocking buffer was removed and 25 l of serial dilutions of sera samples were added. After a 2-h incubation, microplates were washed 4 times with PBS-TWEEN 20 0.05% and 25 l of a 1/5,000 dilution of alkaline phosphatase conjugated F(ab).sub.2 goat anti-mouse IgG (H+L, #115-056-062, Jackson Immunoresearch, Cedarlane, Burlington, ON) in blocking buffer was added. After a 1-h incubation, microplates were washed 4 times and 25 l of p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich Canada Co., Oakville, ON) at 1 mg/ml in carbonate buffer at pH 9.6 was added and further incubated for 30 min. Absorbance was read at 405 nm using a SpectraMax 340 PC plate reader (Molecular Devices, Sunnyvale, CA). All pre-immune bleeds were negative and all post-immune bleeds were very strong (titer above 1/51200) on recombinant protein.

(162) Generation of Hybridomas

(163) Mice received a final boost of 100 g of recombinant EGFRvIII protein and their spleen was harvested 3 to 4 days later. All manipulations were done under sterile conditions. Spleen cells were harvested in Iscove's Modified Dulbecco's medium (IMDM, Gibco Cat. #31980-030) and fused to NS0 myeloma cell line using electrofusion protocol.

(164) Electrofusion Protocol

(165) Spleen cells and myeloma cells were washed separately in IMDM. Cells were washed in Isoosmolar buffer (Eppendorf cat #4308070536), then in Cytofusion Medium C (BTX cat #47-0001). Myeloma and lymphocytes were mixed together at a 1:1 ratio and fused using an ECM 2001 Cell Fusion System (BTX, Harvard Bioscience Inc.) following manufacturer's instructions.

(166) Following fusion, cells were suspended at a concentration of 2-410.sup.5 input myeloma cells per ml in HAT selection medium (IMDM containing 20% heat inactivated FBS, penicillin-streptomycin (Sigma Cat #P7539), 1 ng/ml mouse IL-6 (Biolegend Cat #575706), HAT media supplement (Sigma Cat #H0262) and L-glutamine (Hy-Clone Cat #SH30034.01) and incubated at 37 C., 5% CO.sub.2. The next day, hybridoma cells were washed and suspended at a concentration of 2-510.sup.5 input myeloma cells per ml in semi-solid medium D (StemCell Technologies Cat. #03804) supplemented with 5% heat inactivated FBS, 1 ng/ml mouse IL-6 and 10 g/ml FITC-F(ab).sub.2 Goat anti-mouse IgG Fc gamma specific (Jackson #115-096-071). The cell mixture was plated in Omnitray dish (Nunc cat #242811) and further incubated for 6-7 days at 37 C., 5% CO.sub.2. Fluorescent secretor clones were then transferred using a mammalian cell clone picker (ClonepixFL, Molecular Devices) into sterile 96-w plates (Costar #3595) containing 200 l of IMDM supplemented with 20% heat inactivated FBS, penicillin-streptomycin, 1 ng/ml mouse IL-6, HT media supplement (Sigma Cat #H0137) and L-glutamine and incubated for 2-3 days at 37 C., 5% CO.sub.2.

(167) Five thousand (5000) hybridoma supernatants from seven (7) fusion experiments were screened by ELISA using recombinant EGFRvIII or EGFR wild-type proteins to detect specific binders. To this end, half-area 96-well plates (Costar #3690) were coated with 25 l per well of immunogen at 5 g/ml in PBS and incubated overnight at 4 C. Microplates were washed three times in PBS and blocked for 30 min with PBS containing 1% bovine serum albumin (BSA, Sigma Cat #A7030). Blocking buffer was removed and 25 l of hybridoma supernatant were added. After a 2-h incubation, microplates were washed 4 times with PBS-TWEEN 20 0.05% and 25 l of a 1/5,000 dilution of alkaline phosphatase conjugated F(ab).sub.2 goat anti-mouse IgG (Fc specific, #115-056-071, Jackson Immunoresearch, Cedarlane, Burlington, ON) in blocking buffer was added. After a 1-h incubation, microplates were washed 4 times and 25 l of p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich Canada Co., Oakville, ON) at 1 mg/ml in carbonate buffer at pH 9.6 was added and further incubated for one hour at 37 C. Absorbance was read at 405 nm using a SpectraMax 340 PC plate reader (Molecular Devices, Sunnyvale, CA).

(168) ELISA positive antibodies were selected and further characterized by flow cytometry on U87MG cells overexpressing wild-type human EGFR (U87MG-EGFR WT) or human EGFRvIII (U87MG-EGFRvIII) to confirm their specificity. To this end, 15-ml supernatant from each positive clone was produced.

(169) In order to compare our results with previous studies, additional monoclonal antibodies were used including the 13.1.2 antibody which is specific to EGFRvIII mutation (Hamblett K. J, et al., 2015; U.S. Pat. No. 7,736,644) and the 225 monoclonal antibody which is a murine antibody recognizing both wild-type human EGFR and human EGFRvIII proteins (Mendelson et al.; 2015).

Example 2: Cell Surface Binding by Flow Cytometry

(170) For specificity analysis, several cell lines were used including human glioblastoma cell lines U87MG overexpressing wild-type human EGFR (a.k.a., U87MG-EGFR WT or U87 WT) and U87MG overexpressing human EGFRvIII (42-7 deletion mutation of EGFR a.k.a. U87MG-EGFRvIII or U87vIII).

(171) The binding properties of the anti-EGFRvIII monoclonal antibodies selected in Example 1 were assessed by flow cytometry on human glioblastoma cell lines U87MG overexpressing wild-type EGFR and U87MG overexpressing EGFRvIII mutation.

(172) Briefly, cells overexpressing full length wt EGFR or EGFRvIII were obtained from the laboratory of W. Cavanee (Ludwig Institute for Cancer Research, University of California at San Diego). Cells were grown in DMEM high glucose medium containing 10% FBS and 400 g/ml G418. Prior to analysis, cells were plated such that they were not more than 80% confluent on the day of analysis. Unless otherwise stated, all media are kept are 4 C. and all incubations are performed on wet ice. Cells were washed in PBS and harvested by the addition of cell dissociation buffer (Sigma), centrifuged and resuspended in complete medium at a cell density of 210.sup.6 cells/mL. Fifty L/well of cells are distributed in a polypropylene v-bottom 96 well plate and equal volume of hybridoma supernatant were added and incubated for 2 hours. Cells were washed twice by centrifugation and further incubated with a FITC labeled F(ab).sub.2 goat anti-mouse antibody (Fc specific, #115-096-071, Jackson Immunoresearch, Cedarlane, Burlington, ON) for an hour. Cells were washed and resuspended in medium containing Propidium iodide to exclude dead cells from analysis. Samples were filtered through a 60 m nylon mesh filter plate (Millipore, Ireland) to remove cell aggregates. Flow cytometry analyses were performed on 2,000 viable single-cells events gated on forward scattering, side scattering parameters and propidium iodide dye exclusion using a BD-LSR Fortessa flow cytometer (Becton-Dickinson Biosciences, CA, USA) and a standard filter set using BD FACSDiva acquisition software, according to manufacturer's instructions.

(173) Cells were stained with either negative control anti-GFP 3E6 monoclonal antibody supernatant (open histograms) or tested hybridoma supernatant (grey histograms). Specific binding was reflected by the increase in the mean fluorescent intensity of antibody binding to U87 cells expressing EGFRvIII but not wild-type EGFR.

(174) Out of the 36 positive cell based binding antibodies derived from 7 independent fusion experiments, we chose to further study three hybridoma supernatants, whose binding was found to be specific for EGFRvIII overexpressing U87MG cells, including F265-5B7 (FIG. 1A) referred to herein also as 5B7, F269-3D12 (FIG. 1B) referred to herein also as 3D12 and F271-1D2 (FIG. 1C) referred to herein also as 1D2. Data obtained for the 225 antibody (ATCC HB-8508) and the 13.1.2 antibody are shown in FIGS. 1D and 1E respectively.

Example 3: Evaluation of Binding on Purified Denatured Antigen

(175) To evaluate if monoclonal antibodies bind to a conformational epitope, an ELISA analysis on native and denatured recombinant human wild-type EGFR and EGFRvIII proteins were performed. The 13.1.2 antibody) and the 225 antibody were used as controls. Antigens at 1-2 mg/ml were denatured by incubation at 95 C. for 5 min in PBS containing DTT at 40 mM final concentration. They were then incubated on ice for 5 min and diluted at their final coating concentration for ELISA purpose.

(176) mAbs were purified using HiTrap ProteinG HP 1 mL columns GE Healthcare cat no. 17-0404-01 and desalted using Zeba-spin desalting columns 5 mL (Pierce) pre-equilibrated in PBS and filter sterilized through 0.22 UM membrane (Millipore). The final concentration of the antibody solutions was determined using a Nano-drop 2000 (ThermoScientific), using IgG as sample type. ELISA was performed as described above (serum titer determination) using 25 l of mAb supernatant (Exp 1) or purified mAb at 1 g/ml (Exp 2).

(177) Table 1 shows ELISA results (n=2) of different mAb clones assessed on recombinant human EGFRvIII or wild-type EGFR, in native or denatured conditions. As expected, the 225 antibody binds to both wild-type EGFR and EGFRvIII under native conditions only. The 13.1.2 antibody binds to EGFRvIII in native and denatured conformations, but not to EGFR wild-type native or denatured. All other mAbs binds to EGFRvIII in native and denatured conformations.

(178) TABLE-US-00002 TABLE 1 Binding on native or denatured recombinant proteins by ELISA EGFRvIII EGFRwt Native Denatured Native Denatured Clone Exp 1 Exp 2 Exp 1 Exp 2 Exp 1 Exp 2 Exp 1 Exp 2 5B7 1.144 0.874 1.674 0.623 0.008 0.002 0.046 0.000 3D12 1.173 1.125 1.859 0.517 0.095 0.011 0.045 0.001 1D2 1.491 0.935 2.553 1.054 0.002 0.004 ND 0.000 225 1.081 1.496 0.001 0.004 1.204 1.320 0.010 0.002 13.1.2 1.097 1.671 1.914 1.705 0.011 0.003 0.003 0.005 mIgG1 13.1.2 1.291 1.487 2.094 1.495 0.009 0.003 0.007 0.002 mIgG2a Neg ctrl ND 0.001 ND 0.002 ND 0.002 ND 0.000 mouse mAb ND: not determined

(179) Selected hybridoma were recloned by limiting dilution to ensure their monoclonality.

Example 4. Epitope Mapping by Yeast Surface Display

(180) The yeast surface display method (Feldhaus M J et al., 2003 Nat Biotechnol. 2003 February; 21(2): 163-70) was used to map the epitopes of our collection of monoclonal antibodies against EGFRvIII. This technique allows cloned protein or peptide of choice to be expressed and displayed at cell surface through covalent linkage to cell wall. The displayed protein/peptide can be interrogated for antibody binding.

(181) A total of 36 different human EGFRvIII fragments of variable size from 10 to 414 as indicated in FIG. 3 were cloned into the pPNL6 vector (obtained from The Pacific Northwest National Laboratory, USA) as fusion proteins to be expressed and displayed as Aga2-HA-hEGFRvIII-MYC. The displayed human EGFRvIII fragments were used to identify the smallest fragment required for the binding of each anti-EGFRvIII monoclonal antibodies.

(182) Assessment of the binding of anti-EGFRvIII monoclonal antibodies to the fusion proteins expressed on yeast cell surface was done by flow cytometry analysis. Yeast cells were labeled with both the anti-EGFRvIII monoclonal antibody and chicken anti-Myc antibody the latter being used to monitor the level of expression of the fusion protein. Following a wash step, binding of the primary antibodies is probed by a two-color indirect fluorescence labeling using a specific mouse and chicken secondary antibodies for each of the primary antibody respectively.

(183) The anti-EGFRvIII monoclonal antibodies were binding with similar signal intensities to both full length hEGFRvIII protein and small peptides of the same protein, as well to both native and heat denatured yeast displayed antigen fragments, suggesting that the epitopes for the mAbs are contained within a continuous peptide fragment (linear).

(184) The results presented in FIG. 3 illustrate the binding properties of the anti-EGFRvIII monoclonal antibodies to various fragments of the EGFRvIII and wild-type EGFR protein cells displayed on yeast cells.

(185) Based on the results presented in FIG. 3, the smallest EGFRvIII fragment (peptide) that the anti-EGFRvIII antibodies were able to bind was identified (Table 2). Two different epitope bins were identified: 1) aa 1-18 recognized by 1D2 as well as the 13.1.2 control mAb, 2) aa 15-37 recognized by 5B7 and 3D12. Surprisingly, despite the fact that all antibodies were capable of binding specifically to EGFRvIII expressing cells, the 5B7 and 3D12 monoclonal antibodies bind to an epitope that doesn't encompass the spliced junction neoepitope (aa 5-7) found exclusively in EGFRvIII. Despite the absence of binding on U87MG-EGFR WT expressing cells. 5B7 did bind slightly to yeast cells engineered to express EGFR WT fragments 266-482 (FIG. 3).

(186) TABLE-US-00003 TABLE 2 Summary of epitope binning Epitope Containing mAb Clone Name EGFRvIII Fragment 5B7 aa 15-37 3D12 aa 15-37 1D2 aa 1-18 hFc-13.1.2 aa 1-18 225 aa 43-456

Example 5. Fine Epitope Mapping Using Yeast Surface Display

(187) To further characterize mAb epitopes within the EGFRvIII 15-37 region, an alanine scan of this region was performed, and modified fragments were expressed at the surface of the yeast. SEQ ID NO:5 shows the amino acid sequence of the EGFRvIII 15-37 fragment, where each underlined amino acid was mutated to alanine. The resulting DNAs were expressed at the surface of the yeast and each anti-EGFRvIII mAbs was tested on the corresponding yeast mutant strain by flow cytometry analysis. The original Ala19 and Ala22 were not mutated. Thus, this assay determined the contribution of each amino acid(s) in mAb binding in comparison to the original wild-type fragment (SEQ ID NO:5) which is attributed the value of 100%.

(188) Table 3 shows the results obtained in flow cytometry analysis for the 5B7 and 3D12 monoclonal antibodies. Results obtained are in line with the results of FIG. 3 i.e. binding of mAbs in bin 15-37 are strongly inhibited by mutation of at least one amino acid residue between position 15 and 19. This assay shows that the binding of the 5B7 monoclonal antibody is compromised by mutation of Val17, Arg18, Cys20, Gly21, Asp23 and Cys35. The binding of the 3D12 monoclonal antibody is strongly inhibited by mutation of Arg18, Cys20, Gly21, Tyr25, Glu26, Gly31 and Cys35, and weakened by Glu29 and Arg33 mutation to Alanine.

(189) TABLE-US-00004 TABLE 3 Flow cytometry evaluation of mAb binding to yeast expressing mutated amino acid within fragment 15-37 of EGFRvIII. Data represent the % of binding of mAb on the yeast displaying the mutated sequence compared to the binding on the yeast displaying the wild-type sequence, normalized to the Myc-tag expression. Amino acid mutated to mAbs Corresponding Ala 5B7-2 3D12-2 peptide sequence Ser15 91.6 90.2 SEQ ID: 48 Cys16 240.2 170.7 SEQ ID: 49 Val17 0.3 116.2 SEQ ID: 50 Arg18 0.2 0.1 SEQ ID: 51 Ala19* ND ND Cys20 0.4 0.2 SEQ ID: 52 Gly21 0.1 0.0 SEQ ID: 53 Ala22* ND ND Asp23 0.8 62.4 SEQ ID: 54 Ser24 230.1 232.8 SEQ ID: 55 Tyr25 192.5 3.5 SEQ ID: 56 Glu26 140.1 1.4 SEQ ID: 57 Met27 111.4 75.3 SEQ ID: 58 Glu28 78.5 71.0 SEQ ID: 59 Glu29 109.7 47.7 SEQ ID: 60 Asp30 105.6 78.5 SEQ ID: 61 Gly31 98.9 10.7 SEQ ID: 62 Val32 101.1 93.8 SEQ ID: 63 Arg 33 95.8 22.1 SEQ ID: 64 Lys34 192.7 137.0 SEQ ID: 65 Cys35 1.5 0.1 SEQ ID: 66 Lys36 87.2 95.1 SEQ ID: 67 Lys37 143.3 162.3 SEQ ID: 68 *Original Ala 19 and Ala 22 were not mutated Legend Values between 0% and 15%: No binding Values between 16% and 59%: Partial binding Values at or above 60%: Complete binding

Example 6: Antibody Sequencing

(190) The sequence of the VH and VL regions of the anti-EGFRvIII antibodies 5B7, 3D12 and 1D2 were analyzed.

(191) Briefly, total RNA was extracted from hybridoma clones (Qiagen, RNEasy) and reverse transcribed into cDNA (SuperScript, ThermoFisher Scientific, Waltham, MA, USA). DNA encoding VH and VL domains was PCR amplified (Platinum Taq or equivalent) using mixtures of degenerate forward primers annealing in FR1 and a single reverse primer annealing in CH1 (Novagen/EMD Millipore cat. no 69831-3). The resulting amplicons were sequenced using the Sanger method on an ABI 3730xl instrument or were determined using 2250 bp reads on an 10 Illumina MiSeq instrument.

(192) Sequences of the VH and VL regions as well as the CDR regions are shown in the Sequence Table section. Analysis of the sequence for a consensus binding sequence of the CDR 1-3 regions of the VH and VL chains was conducted using Kabat numbering scheme (Kabat et al 1992, Johnson et al 2004). The results of this analysis indicated that 5B7, 3D12 and 1D2 monoclonal antibodies have unique VH and VL CDRs.

Example 7: EGFRvIII CAR-T Design and Cloning

(193) To generate a chimeric antigen receptor sequence using the EGFRvIII-specific antibody sequences (as described above), amino acid sequences based on the variable regions of the 5B7, 3D12, 1D2 monoclonal antibodies were assembled into single chain variable fragments (scFv) containing their respective heavy chain, a (GGGGS).sub.3 (SEQ ID NO: 89) linker sequence (although any appropriate linker could be used), followed by the light chain in silico. An amino acid sequence compatible with a restriction site was included at the beginning of the linker sequence to allow later recombination as needed. In addition, a spacer sequence was attached to the 3 end of the sequence. In the present case, a hinge domain derived from mouse CD8 was used, but this sequence can also be substituted for other spacer domains as described herein.

(194) FIG. 4 illustrates the synthetic assembly of 5B7 amino acids (SEQ ID NO: 30) for insertion in CAR vector to generate EGFRvIII 5B7 CAR (SEQ ID NO:32). Sequence shows the heavy chain portion (underlined), amino acids corresponding to a restriction site (*), linker (double underlined), the light chain portion (underlined), and the mouse CD8 hinge. The 3D12 and 1D2 sequences were assembled similarly (SEQ ID NO:33 and SEQ ID NO:35 respectively) to generate EGFRvIII 3D12 CAR (SEQ ID NO:34) and EGFRvIII 1D2 CAR (SEQ ID NO:36) respectively.

(195) After in silico assembly, sequences were reverse translated into DNA using a standard human codon usage table via online tool available to the public (Chojnacki, S. et al., Nucl Acid Res. 45 (W1): W550-553, 2017). Specific DNA sequences for various restriction sites were avoided and appropriate DNA linkers were added to the ends of the DNA to allow insertion of the sequence into a modular CAR vector using a scarless cloning strategy based on a Type IIS restriction enzyme (Bbsl). DNA was then synthesized by Integrated DNA Technologies in the form of a linear double stranded gene fragment.

(196) The DNA sequence of the 5B7 scFv and hinge as synthesized is presented in SEQ ID NO: 31. Sequence shows heavy chain (underlined), restriction sites (nucleotide 5 to 10, 412 to 418 and 1039 to 1045), linker (419 to 457), light chain (underlined), and mouse CD8 hinge (twice underlined). Sticky end sequences for scarless insertion via type IIs cloning are shown in bold.

(197) This sequence was then inserted into our modular CAR vector (SEQ ID NO:37, FIG. 5A). The modular CAR vector comprises nucleic acid sequence encoding a human CD28 signal peptide (nucleotide 1 to 50), restriction sites (nucleotides 57 to 63 and 65 to 70) including sticky end sequences shown in black, filler (irrelevant sequence), a human CD28 transmembrane domain (nucleotide 84 to 165), a human CD28 intracellular transduction domain (nucleotide 167 to 294), and a human CD3 zeta intracellular transduction domain (nucleotide 295 to 642). Linker sequences for scarless insertion via type IIs cloning are shown in bold.

(198) Certain truncations of the CAR sequences especially truncations in the scFv portion (as exemplified in SEQ ID NO:78) can retain similar activity while removing some backbone elements of the antibody heavy or light chain. Exemplary data is provided to show that a CAR plasmid containing truncated versions of both the VH and VL elements of 1D2-CAR has minimal effect on the ability of CAR-transduced T cells to respond to target cells as measured using electroporated Jurkat cells exposed to varying doses of EGFRvIII+ target cells (FIG. 5B and FIG. 5C).

(199) Sequences were combined into a modular plasmid vector which also contained a human EF1a promoter, P2A-GFP marker and Lentiviral transfer elements. Construction was confirmed using clonal sequencing in Escherichia coli bacteria, and plasmids were isolated by standard technique.

(200) Alternatively, the scFv sequences described herein can be inserted into vectors for expression of bi-specific T-cell engagers, bispecific killer cell engagers or trispecific killer cell engagers (FIG. 5D).

Example 8: Detection of CAR Expression on Transduced Cells

(201) Two strategies were employed to detect the expression of CAR molecule(s) on transduced immune cells. As a research strategy, the green fluorescence protein (GFP) was co-expressed with the EGFRvIII CAR and used as a surrogate for monitoring CAR expression in transduced cells.

(202) Briefly, the Jurkat human CD8+ T-cell line was transiently transfected with the EGFRvIII construct which also expressed GFP using a 2A self-cleaving peptide based multi-gene expressing system. Expression of EGFP was quantified using flow cytometry (FIG. 6A) and the surface expression of CAR was detected by flow cytometry using a fluorescent tagged antibody that recognize murine IgG (Goat Anti-Mouse IgG H&L [Alexa Fluor 594]; Jackson Immunoresearch) (FIG. 6B).

(203) The flow cytometry analysis showed an increased level of GFP expression in EGFRvIII CAR (EGFRvIII 5B7 CAR (a.k.a. 5B7), EGFRvIII 3D12 CAR (a.k.a. 3D12) and EGFRvIII 1D2 CAR (a.k.a. 1D2)) transduced cells compared to the control cells (FIG. 6A).

(204) In addition, an antibody recognizing murine IgG sequences within the scFv (GAMFab) was used for direct evaluation of surface expression of the CAR. The EGFRvIII CAR (PSLQC2-5B7 and 1D2) transduced cells were positive for GFP expression and binding of the GAMFab antibody whereas the control cells that were transduced with GFP expressing control plasmid devoid of a CAR construct (PX458) showed GFP expression but no binding of the GAMFab antibody (FIG. 6B).

Example 9: CAR-T Functionality Screening

(205) The in vitro functionality of the EGFRvIII CAR constructs was tested using a novel flow cytometry based high-throughput screening platform developed by the Applicant; which is in some instances referred to as CAR-J assay. In brief, EGFRvIII or control (CD19-targeted) CAR plasmids were electroporated into the Jurkat human CD8+ T-cell line. Cells expressing CAR were then exposed to various target cell lines (with or without EGFRvIII expression) in varying doses and maintained under standard culture conditions. Following 24 hours of co-incubation with target cells, CAR-T cells were examined for cellular activation by flow cytometry via surface expression of the T-cell activation marker CD69. The level of auto-activation (tonic signaling) associated with each CAR was also examined by quantification of the level of CD69 expression on non-stimulated CAR-expressing Jurkat cells or CAR-expressing Jurkat cells incubated with irrelevant target cells. The high-throughput screening method for CAR functionality is summarized in FIG. 7A.

(206) Jurkat cells electroporated with various CAR constructs (X1, X2, 5B7, 3D12 and 1D2) were exposed to varying doses of target cells with (U87vIII, DKMG) or without (U87-MG) specific target expression (FIGS. 7B, C and D). Tonic signaling/auto-activation of each CAR construct was assessed by examining the level of CD69 expression in CAR-expressing (GFP+) cells without additional stimulation (FIG. 7B). The relative increase in expression of CD69 within CAR cells following stimulation with EGFRvIII-expressing target cells is illustrated in FIG. 7C and FIG. 7D.

(207) The results of FIGS. 7B-D indicate that some CAR constructs (X1 and X2) showed high level of activation in the absence of stimulation or when stimulated with target cells that do not express EGFRvIII (U87-MG), indicative of high tonic signaling and therefore would not be suitable for CAR-mediated therapy. The 5B7, 3D12 and 1D2 CAR constructs showed very low level of activation in the absence of stimulation or in response to irrelevant target cells (U87-MG, FIG. 7B) but showed high level of activation in response to specific stimulation with EGFRvIII expressing target cells U87vIII and DKMG (FIGS. 7C and D, respectively). This allowed the selection of the 5B7, 3D12 and 1D2 CAR constructs for further characterization.

(208) In a separate experiment, plasmid expressing CAR targeting EGFRvIII or control plasmids (PX458 expressing GFP but no CAR construct and plasmid expressing CAR targeting wild-type EGFR protein) were electroporated into Jurkat cells. GFP expressing cells and control Jurkat cells (CD45 positive) were then exposed to various target cell lines with (U87vIII) or without (A20 and U87-MG) EGFRvIII expression in varying doses and maintained under standard culture conditions. Following 24 hours of incubation, cells were examined via flow cytometry for GFP expression and cell activation using fluorescent staining against the T-cell activation marker CD69. The level of auto-activation (tonic signaling) associated with each CAR was also examined by quantification of the level of CD69 expression on non-stimulated CAR-expressing Jurkat cells (FIG. 8A).

(209) The baseline level of CD69 expression in unstimulated CAR transduced Jurkat cells is similar to levels seen with unstimulated wild-type Jurkat cells suggesting no detectable tonic signaling in CAR-T cells. Similarly, low level of CD69 expression that were comparable to levels seen with wild-type Jurkat cells was seen with EGFR wild-type and EGFRvIII CAR-T cells when stimulated with human B cell lymphoma cell line A20 that does not express wild-type EGFR or EGFRvIII protein. CAR-T cells targeting wild-type EGFR responded strongly to U87, U87vIII and OVCAR3 cells, which express the wild-type EGFR protein whereas EGFRvIII targeted CAR-T cells responded only to U87vIII cells and not the U87 or OVCAR3 cells (FIG. 8A).

(210) In order to optimize the length of the hinge element that should be integrated in CAR constructs, various constructs were tested using Jurkat activation assay similarly as described above. Briefly, CAR constructs containing either 5B7 scFV (SEQ ID NO:44) or 3D12 scFV (SEQ ID NO: 45) sequence followed by (1) a very long hinge wherein a 17AA poly-glycine linker [(GGGGS) 3GG] (SEQ ID NO: 92) was followed by the 45AA sequence of the hinge domain of the human CD8 protein [L17-CD8h: SEQ ID NO:83], (2) human CD8 hinge alone [45CD8h: SEQ ID NO: 84], (3) a truncated form of CD8-hinge containing C-terminal 35AA [35CD8h: SEQ ID NO:85], (4) a truncated form of CD8-hinge containing C-terminal 15AA [15CD8h: SEQ ID NO:86], or (5) a truncated form of CD8-hinge containing C-terminal 1AA (1CD8h: PLD) were generated. Jurkat cells were then electroporated with vIII-specific constructs with varying hinge length and co-cultured at a 1:1 effector to target ratio with antigen expressing cells (U87III) or antigen-negative cells (U87WT). Results clearly demonstrate that EGFRvIII constructs developed here show high target-specific response across a range of hinge lengths (FIG. 8B).

(211) Based on the in vitro functionality data, EGFRvIII CAR constructs 1D2 and 3D12 were selected for testing for in vivo functionality.

(212) Briefly, athymic nude mice (Jackson Laboratory, Barr Harbor, ME) were injected subcutaneously with 110.sup.6 (FIG. 9A) or 410.sup.6 (FIG. 9B) U87vIII human glioblastoma cells expressing EGFRvIII. CD19 targeted CAR-T was used as control in the studies. On day 5 post tumor cell injection, mice were given 10.sup.5 (FIG. 9A) or 10.sup.6 (FIG. 9B) human primary T cells expressing EGFRvIII 1D2 CAR. Animals were monitored for tumor growth. Tumor size (the length and the width) was measured using a digital vernier caliper. Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter. The tumor volume (meanSEM) is depicted in FIG. 9A and FIG. 9B. FIG. 9C depicts the level of CD8.sup.+ T-cell infiltration in tumors from two animals each from groups receiving EGFRvIII targeted CAR-T and showing good tumor control and irrelevant CAR (CD19 CAR-T).

(213) The results illustrate an increase in tumor growth in all animals receiving irrelevant CD19 CAR-T cells whereas effective tumor control was seen in a subset of animals receiving EGFRvIII CAR-T cells (FIG. 9A and FIG. 9B). EGFRvIII CAR-T treated mice showing good tumor control also showed good CAR-T cell infiltration in the tumor (FIG. 9C).

Example 10: CAR-NK Functional Screening

(214) The human NK-92 cell line (Gong et al; 1994; Leukemia) was used for development and testing of EGFRvIII targeted NK 3D12-CAR cells. The graphic in FIG. 10A represents the generation of EGFRvIII targeted NK92-CAR cells for in vitro testing of tumor cytotoxicity. In brief, EGFRvIII or control (wild-type EGFR protein-targeted) CAR plasmids were electroporated into NK-92 cells (ATCC; Manassas, VA). Cells expressing CAR were then exposed to various target cell lines with (U87vIII) or without (U87) EGFRvIII expression in varying doses and maintained under standard culture conditions. Following 24 hours of co-incubation with target cells, tumor cell killing was assessed by quantifying the release of lactate dehydrogenase (FIG. 10B LDH; a cytosolic enzyme which is release upon cell death) by colorimetry or by using the standard .sup.51Cr-release assay (FIG. 10C). Wild-type NK92 cells devoid of CAR construct was used as negative control.

(215) EGFR WT NK-CAR showed killing of U87 and U87vIII cells both of which overexpress a common epitope present on both EGFR protein isoforms whereas EGFRvIII CAR-NK showed preferential killing of U87vIII cells. The specific cytotoxicity of EGFRvIII targeted NK 3D12-CAR towards EGFRvIII and not the wild-type EGFR protein expressing cells was shown by both LDH (FIG. 10B) and .sup.51Chromium release (FIG. 10C) assays.

(216) The in vivo functionality of the EGFRvIII targeted NK-CARs was also tested using 2 independent experiments using athymic nude mice bearing EGFRvIII expressing U87vIII tumors.

(217) Briefly, athymic nude mice (Jackson Laboratory, Barr Harbor, ME) were injected subcutaneously with 110.sup.6 U87vIII human glioblastoma cells expressing EGFRvIII. On day 2 post tumor cell injection, mice were given either 510.sup.6 wild-type NK92 cells devoid of CAR or NK92 cells expressing EGFRvIII 3D12-CAR-NK (FIG. 11A) or on day 3 post tumor induction animals were given 110.sup.7 EGFRvIII 3D12-CAR-NK or left untreated (FIG. 11B). Animals were monitored for survival (left panels) and tumor growth (right panels). Tumor size (the length and the width) was measured using a digital vernier caliper. Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter.

(218) In both studies, better control of tumor growth and increased survival was seen in animals receiving EGFRvIII NK-CAR compared to animals receiving unmodified NK92 cells or untreated controls (FIG. 11A and FIG. 11B) respectively.

(219) The chimeric antigen receptors (CAR) generated herein may therefore be used to re-direct NK cells to specifically recognize and kill cells expressing EGFRvIII protein.

Example 11: Primary CAR-T Functional Testing In Vitro

(220) Human primary peripheral blood derived T cells were used for confirmation of in vitro and in vivo activity of EGFRvIII targeted 3D12 CARs constructs in the context of primary human immune cells. In brief, EGFRvIII-CAR or control (human CD19-targeted FMC63-CAR) lentivirus was generated using standard production protocols in HEK293 and concentrated using ultracentrifugation. Primary T cells were isolated from donor blood samples using magnetic separation and activated using anti-CD3/CD28 beads. Primary T cells were then transduced with CAR lentivirus and expanded for several days in culture.

(221) In vitro functionality of primary human EGFRvIII-specific CAR-transduced T cells were assessed using a live-fluorescence microscopy approach. Briefly, EGFRvIII targeted CAR-T or mock transduced T cells, wherein no lentiviral construct was introduced into cells handled under similar conditions, were generated as described above. Cells were then placed in co-culture with EGFRvIII-expressing target cells modified to also express a nuclear-localized form of mKate2 fluorescent protein. Co-cultures were monitored constantly over 6 days using the Incucyte automated live fluorescent microscopy device (Sartorius, USA). The relative growth of target cells was then assessed using automated counting of mKate2+ cells (FIG. 12). Data showed that the growth of U87MG overexpressing EGFRvIII cells was efficiently repressed by 3D12 CAR-T compared to mock transfected T-cells.

Example 12: Primary CAR-T Functional Testing In Vivo

(222) The in vivo functionality of the EGFRvIII targeted primary CAR-T cells was also tested using Nod-SCID-IL2R2.sup./ (NSG) mice (Jackson Laboratory, Barr Harbor, ME) bearing EGFRvIII expressing U87vIII tumors. Briefly, mice were subcutaneously injected with 110.sup.6 fluorescently labelled U87-vIII cells. Eight days after tumour cell injection, cryo-preserved CAR-T cells were thawed, washed with PBS, and 110.sup.7 total T cells (with 20-25% CAR transduction) were immediately delivered intra-tumourally, ensuring equal distribution of tumour sizes between groups. Tumour growth was evaluated three times per week using calipers by trained animal technicians blinded to specific treatment groups (FIG. 13A). Primary endpoint was tumour size above 2000 mm.sup.3, with secondary endpoints determined by overall animal health and well-being (FIG. 13B). Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter.

(223) In this study, better control of tumor growth and increased survival was seen in animals receiving EGFRvIII primary CAR-T compared to untreated control animals (FIG. 13A and FIG. 13B) respectively.

Example 13: Bi-Specific Immune Cell Engager Functional Testing

(224) Various experiments were performed to demonstrate the activity of the novel EGFRvIII-specific single chain variable fragment in the context of a bispecific T cell engager. Constructs were generated using synthetic DNA wherein the 3D12 scFV sequence (SEQ ID NO:45) was linked to a previously demonstrated CD3-engaging scFv sequence (SEQ ID NO:82). A plasmid expressing this bi-specific construct was transfected into human embryonic kidney cells (HEK293T) and supernatant was collected after 2 to 4 days in culture. Supernatant from HEK293T were transferred to wells containing Jurkat cells and varying doses of antigen expressing target cells (U87vIII). Target-induced activation in the presence or absence of bispecific T-cell engager was measured by examining the level of CD69 expression using human CD69-specific antibody staining and flow cytometry (FIG. 14). Results showed that only EGFRvIII targeted bi-specific construct could induce high target-specific T-cell activation response compared to a control bi-specific molecule.

(225) In vitro functionality of primary human EGFRvIII-specific bi-specific immune T-cell engager in interaction with primary human T cells were assessed using a live-fluorescence microscopy approach. Briefly, polyclonally expanded human T cells which were allowed to return to a rest state over several weeks in culture were placed in co-culture with EGFRvIII-expressing target cells modified to also express a nuclear-localized form of mKate2 fluorescent protein. Various doses of HEK293T supernatant, or supernatant wherein cells were secreting a control CD19-CD3 targeted or 3D12-CD3 targeted bi-specific immune cell engager were transferred to T-cell target cell co-cultures. Co-cultures were then monitored constantly over 6 days using the Incucyte automated live fluorescent microscopy device (Sartorius, USA). The relative growth of target cells was then assessed using automated counting of mKate2+ cells (FIG. 15). Results demonstrate that 3D12-CD3 bi-specific immune cell engagers can actively induce T-cell mediated repression of EGFRvIII-positive tumour cell growth compared to the bi-specific CD19-CD3 control molecule or mock supernatant.

(226) The embodiments and examples described herein are illustrative and are not meant to limit the scope of the disclosure as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Citations listed in the present application are incorporated herein by reference.

REFERENCES

(227) All patents, patent applications and publications referred to throughout the application are incorporated herein by reference. Andris-Widhopf, J., et al. Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. Cold Spring Harbor protocols, 2011(9). Bird et al. Science 242:423-426(1988) Chojnacki, S. et al., Nucl Acid Res. 45 (W1): W550-553(2017) Chothia C, Lesk A M. Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol. August 20: 196(4): 901-17(1987). Feldhaus M J et al., 2003 Nat Biotechnol. February; 21(2): 163-70(2003). Gacerez, A. T. et al., J Cell Physiol. 231(12): 2590-2598(2016), Gan H. K., Anna N. Cvrljevic, Terrance G. Johns. The epidermal growth factor receptor variant III (EGFRvIII): where wild things are altered. FEBS Journal 280; 5350-5370(2013). Gong et al; Leukemia, 8(4): 652-658(1994) Hamblett K. J, et al., Molecular Cancer Therapeutics, Vol. 14(7), pp. 1614-24(2015). Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883(1988). Jones, P. T. et al., Nature 321:522-525(1986). Johnson G, Wu T T. The Kabat database and a bioinformatics example. Methods Mol Biol., 248:11-25(2004). Kabat E A, Wu T T. Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. J Immunol., 147:1709-19(1991). Lefranc, M.-P., The Immunologist, 7, 132-136(1999). Mendelsohn J, Prewett M, Rockwell P, Goldstein N I. CCR 20th anniversary commentary: a chimeric antibody, C225, inhibits EGFR activation and tumor growth. Clin Cancer Res. 21(2): 227-9(2015). Queen C, Schneider W P, Selick H E, Payne P W, Landolfi N F, Duncan J F, Avdalovic N M, Levitt M, Junghans R P, Waldmann T A, Proc Natl Acad Sci USA 86, 10029-10033(1989). Riechmann, L. et al., Nature 332:323-327(1988). Sadelain, M. et al. Cancer Discovery, 3(4): 388-98, (2013). Sblattero & Bradbury, 2000, Nature Biotechnology, 18(1): 75-80, (2000). Schaefer, J. V, et al. Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly. (R. Kontermann & S. Dbel, Eds.), (2010). Tatusova, et al. FEMS Microbiol Lett. 174:247-250 (1999). Tempest P R, Bremmer P, Lambert M, Taylor G, Furze J M, Carr F J, Harris W J Biotechnology 9, 266-271 (1991). 5 Tsurushita N, Hinton, RP, Kumar S Design of humanized antibodies: From anti-Tac to Zenapax. Methods 36, 69-83 (2005). U.S. Pat. No. 7,736,644. Wang, X et al., Molecular Therapy-Oncolytics, 3:16015, 2016 Ward et al., Nature 341:544-546 (1989). Zhang, C. et al., Biomarker Research, 5:22 (2017).

(228) TABLE-US-00005 SEQUENCETABLE CDRsaregenerallyindicatedinboldand/orunderlined Seq. ID Description Sequence 1 Wildtype CTGGAGGAAAAGAAAGTTTGCCAAGGCACGAGTAACAAGCTCACGCAGTT humanEGFR GGGCACTTTTGAAGATCATTTTCTCAGCCTCCAGAGGATGTTCAATAACT ectodomain GTGAGGTGGTCCTTGGGAATTTGGAAATTACCTATGTGCAGAGGAATTAT cDNA GATCTTTCCTTCTTAAAGACCATCCAGGAGGTGGCTGGTTATGTCCTCAT sequence TGCCCTCAACACAGTGGAGCGAATTCCTTTGGAAAACCTGCAGATCATCA GAGGAAATATGTACTACGAAAATTCCTATGCCTTAGCAGTCTTATCTAAC TATGATGCAAATAAAACCGGACTGAAGGAGCTGCCCATGAGAAATTTACA GGAAATCCTGCATGGCGCCGTGCGGTTCAGCAACAACCCTGCCCTGTGCA ACGTGGAGAGCATCCAGTGGCGGGACATAGTCAGCAGTGACTTTCTCAGC AACATGTCGATGGACTTCCAGAACCACCTGGGCAGCTGCCAAAAGTGTGA TCCAAGCTGTCCCAATGGGAGCTGCTGGGGTGCAGGAGAGGAGAACTGCC AGAAACTGACCAAAATCATCTGTGCCCAGCAGTGCTCCGGGCGCTGCCGT GGCAAGTCCCCCAGTGACTGCTGCCACAACCAGTGTGCTGCAGGCTGCAC AGGCCCCCGGGAGAGCGACTGCCTGGTCTGCCGCAAATTCCGAGACGAAG CCACGTGCAAGGACACCTGCCCCCCACTCATGCTCTACAACCCCACCACG TACCAGATGGATGTGAACCCCGAGGGCAAATACAGCTTTGGTGCCACCTG CGTGAAGAAGTGTCCCCGTAATTATGTGGTGACAGATCACGGCTCGTGCG TCCGAGCCTGTGGGGCCGACAGCTATGAGATGGAGGAAGACGGCGTCCGC AAGTGTAAGAAGTGCGAAGGGCCTTGCCGCAAAGTGTGTAACGGAATAGG TATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAAC ACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTG GCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGA ACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTC AGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAA ATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGT CAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTG ATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTAT AAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATG CCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTC TCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCT TCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGT GCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGG GGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTG CGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCT GGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGC ACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCC TAAGATCCCGTCC 2 HumanEGFR LEEKKVCQGTSNKLTQLGTFEDHELSLQRMENNCEVVLGNLEITYVQRNY ectodomain DLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSN aminoacid YDANKTGLKELPMRNLQEILHGAVRESNNPALCNVESIQWRDIVSSDELS sequence NMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCR GKSPSDCCHNQCAAGCTGPRESDCLVCRKERDEATCKDTCPPLMLYNPTT YQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVR KCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPV AFRGDSFTHTPPLDPQELDILKTVKEITGELLIQAWPENRTDLHAFENLE IIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANT INWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCV SCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGR GPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNC TYGCTGPGLEGCPTNGPKIPS 3 Human CTGGAAGAGAAGAAAGGCAACTACGTCGTGACCGACCACGGCAGCTGTGT EGFRvIII GCGGGCTTGTGGCGCCGATAGCTACGAGATGGAAGAGGACGGCGTGCGGA ectodomain AGTGCAAGAAGTGCGAGGGCCCCTGCCGGAAAGTGTGCAACGGCATCGGC cDNA ATCGGAGAGTTCAAGGACAGCCTGAGCATCAACGCCACCAACATCAAGCA sequence CTTCAAGAACTGCACCAGCATCAGCGGCGACCTGCACATCCTGCCCGTGG (nucleotides1- CCTTTAGAGGCGACAGCTTCACCCACACCCCCCCACTGGACCCCCAGGAA 996) CTGGACATCCTGAAAACCGTGAAAGAGATCACCGGCTTTCTGCTGATTCA GGCCTGGCCCGAGAACCGGACAGACCTGCACGCCTTCGAGAACCTGGAAA TCATCCGGGGCAGGACCAAGCAGCACGGCCAGTTTTCTCTGGCCGTGGTG TCCCTGAACATCACCAGCCTGGGCCTGCGGAGCCTGAAAGAAATCAGCGA CGGCGACGTGATCATCTCCGGCAACAAGAACCTGTGCTACGCCAACACCA TCAACTGGAAGAAGCTGTTCGGCACCTCCGGCCAGAAAACAAAGATCATC AGCAACCGGGGCGAGAACAGCTGCAAGGCCACAGGACAAGTGTGCCACGC CCTGTGTAGCCCTGAGGGCTGTTGGGGACCCGAGCCCAGAGATTGCGTGT CCTGCAGAAACGTGTCCCGGGGCAGAGAATGCGTGGACAAGTGCAACCTG CTGGAAGGCGAGCCCCGCGAGTTCGTGGAAAACAGCGAGTGCATCCAGTG CCACCCCGAGTGTCTGCCCCAGGCCATGAACATTACCTGCACCGGCAGAG GCCCCGACAACTGTATCCAGTGCGCCCACTACATCGACGGCCCCCACTGC GTGAAAACCTGTCCTGCTGGCGTGATGGGAGAGAACAACACCCTCGTGTG GAAGTACGCCGACGCCGGCCATGTGTGCCACCTGTGTCACCCCAAT 4 Human LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIG EGFRvIII IGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQE ectodomain LDILKTVKEITGELLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVV aminoacid SLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKII sequence SNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNL (aminoacids1- LEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHC 332) VKTCPAGVMGENNTLVWKYADAGHVCHLCHPN 5 Human SCVRACGADSYEMEEDGVRKCKK EGFRvIII aminoacid residues15to 37 6 5B7lightchain DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPK variableregion RLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFP (CDRsinbold) QTFGGGTKLEIK 7 5B7CDRL1 KSSQSLLDSDGKTYLN 8 5B7CDRL2 LVSKLDS 9 5B7CDRL3 WQGTHEPQT 10 5B7heavy QIQLVQSGPELKKPGETVMISCKASGYSFTNYGMNWVKQAPEKDLKWMGW chainvariable INTYTGESRYVDEFKGRFAFSLETSVSIVYLKINNLKNEDMATYFCARGP region NFDVWGTGTTVTVSS 11 5B7CDRH1 NYGMN 12 5B7CDRH2 WINTYTGESRYVDEFKG 13 5B7CDRH3 GPNFDV 14 3D12light EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIY chainvariable GTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFG region SGTKLEIK 15 3D12CDRL1 SVSSSISSSNLH 16 3D12CDRL2 GTSNLAS 17 3D12CDRL3 QQWSSYPLT 18 3D12heavy EVQLQQSGPELVKPGSSVRISCKASGYTFTDYNMDWVKQSHGKSLEWIGT chainvariable INPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARVR region QLGLWFAYWGQGTLVTVSA 19 3D12CDRH1 DYNMD 20 3D12CDRH2 TINPNNGGTSYNQKFKG 21 3D12CDRH3 VRQLGLWFAY 22 1D2lightchain DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPK variableregion LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVP WTFGGGTKLEIK 23 1D2CDRL1 RSSQSLVHSDGNTYLH 24 1D2CDRL2 KVSNRFS 25 1D2CDRL3 SQSTHVPWT 26 1D2heavy DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMG chainvariable YIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYCTRSP region YFDVWGTGTTVTVSSA 27 1D2CDRH1 SGYSWH 28 1D2CDRH2 YIHYSGSTNYNPSLKS 29 1D2CDRH3 SPYFDV 30 5B7CAR QIQLVQSGPELKKPGETVMISCKASGYSFTNYGMNWVKQAPEKDLKWMGW constructamino INTYTGESRYVDEFKGRFAFSLETSVSIVYLKINNLKNEDMATYFCARGP acidsequence NFDVWGTGTTVTVSSAKTTAPSVYPLAPGSLGGTGGGSGGGGSGGGGSDV (withlinkerand VMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRL CD8hinge) IYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHEPOT FGGGTKLEIKRADAAPTVSIFPPSSKLGVISNSVMYFSSVVPVLQKVNST TTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFA 31 5B7CAR GACAGAAGACCTAGGACAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGA construct AGAAGCCTGGAGAGACAGTCATGATCTCCTGCAAGGCTTCTGGGTATTCC nucleotide TTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGAAAAGGATTT sequence AAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGTCAAGATATGTTG ATGAATTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGTTAGTATT GTCTATTTGAAGATCAACAACCTCAAAAATGAGGACATGGCTACATATTT CTGTGCAAGAGGGCCTAATTTCGATGTCTGGGGCACAGGGACCACGGTCA CTGTCTCCTCAGCCAAAACAACAGCCCCATCCGTCTATCCCCTGGCCCCT GGAAGCTTGGGAGGTACCGGCGGAAGTGGAGGCGGAGGATCTGGCGGCGG AGGATCCGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCA TTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGAT AGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTC TCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTG ACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGC AGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACA TTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTG ATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAAGCTTGGGGTC ATCAGCAACTCGGTGATGTACTTCAGTTCTGTCGTGCCAGTCCTTCAGAA AGTGAACTCTACTACTACCAAGCCAGTGCTGCGAACTCCCTCACCTGTGC ACCCTACCGGGACATCTCAGCCCCAAAGACCAGAAGATTGTCGGCCCCGT GGCTCAGTGAAGGGGACCGGATTGGACTTCGCCCCTTGGGTCTTCGGTAG GG 32 5B7CAR MLRLLLALNLEPSIQVTGQIQLVQSGPELKKPGETVMISCKASGYSFTNY constructwith GMNWVKQAPEKDLKWMGWINTYTGESRYVDEFKGRFAFSLETSVSIVYLK CD28 INNLKNEDMATYFCARGPNFDVWGTGTTVTVSSAKTTAPSVYPLAPGSLG intracellular GTGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDG transduction KTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVE domain(ITD) AEDLGVYYCWQGTHEPQTFGGGTKLEIKRADAAPTVSIFPPSSKLGVISN andCD3zeta SVMYFSSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSV ITD(signal KGTGLDFAPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD peptide YMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASLRVKFSRSADAPAYQQGQ underlined) NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 33 3D12CAR EVQLQQSGPELVKPGSSVRISCKASGYTFTDYNMDWVKQSHGKSLEWIGT constructamino INPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARVR acidsequence QLGLWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSLGGTGGGSGGGGSGGG (withlinkerand GSEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPW CD8hinge) IYGTSNLASGVPVRESGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLT FGSGTKLEIKRADAAPTVSIFPPSSKLGVISNSVMYFSSVVPVLQKUNST TTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFA 34 3D12CAR MLRLLLALNLEPSIQVTGEVQLQQSGPELVKPGSSVRISCKASGYTFTDY constructwith NMDWVKQSHGKSLEWIGTINPNNGGTSYNQKFKGKATLTVDKSSSTAYME CD28ITDand LRSLTSEDSAVYYCARVRQLGLWFAYWGQGTLVTVSAAKTTPPSVYPLAP CD3zetaITD GSLGGTGGGSGGGGSGGGGSEIVLTQSPALMAASPGEKVTITCSVSSSIS (signalpeptide SSNLHWYQQKSETSPKPWIYGTSNLASGVPVRESGSGSGTSYSLTISSME underlined) AEDAATYYCQQWSSYPLTFGSGTKLEIKRADAAPTVSIFPPSSKLGVISN SVMYFSSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSV KGTGLDFAPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD YMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASLRVKESRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 35 1D2CAR DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMG constructamino YIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYCTRSP acidsequence YFDVWGTGTTVTVSSAKTTPPSVYPLAPGSLGTGGGSGGGGSGGGGSDVV (withlinkerand MTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLI CD8hinge) YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTF GGGTKLEIKRADAAPTVSIFPPSSKLGVISNSVMYFSSVVPVLQKVNSTT TKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFA 36 1D2CAR MLRLLLALNLEPSIQVTGDVQLQESGPDLVKPSQSLSLTCTVTGYSITSG constructwith YSWHWIRQFPGNKLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQ CD28ITDand LSSVTTEDTATYYCTRSPYFDVWGTGTTVTVSSAKTTPPSVYPLAPGSLG CD3zetaITD TGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGN (signalpeptide TYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSGSGTDETLKISRVEA underlined) EDLGVYFCSQSTHVPWTFGGGTKLEIKRADAAPTVSIFPPSSKLGVISNS VMYFSSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVK GTGLDFAPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY MNMTPRRPGPTRKHYQPYAPPRDFAAYRSASLRVKESRSADAPAYQQGON QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 37 DNAsequence ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAAC formodular AGGAGGGTCTTCGAGAAGACCTCCTTCTAAGCCCTTTTGGGTGCTGGTGG CARvector TGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTT (without ATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTA filler/insert) CATGAACATGACTCCCAGGCGGCCCGGACCCACCCGCAAGCATTACCAGC CCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGCTAGCCTGAGA GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCTCGAG 38 5B7Heavy CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGAC chainvariable AGTCATGATCTCCTGCAAGGCTTCTGGGTATTCCTTCACAAACTATGGAA region TGAACTGGGTGAAGCAGGCTCCAGAAAAGGATTTAAAGTGGATGGGCTGG nucleotide ATAAACACCTACACTGGAGAGTCAAGATATGTTGATGAATTCAAGGGACG sequence GTTTGCCTTCTCTTTGGAAACCTCTGTTAGTATTGTCTATTTGAAGATCA ACAACCTCAAAAATGAGGACATGGCTACATATTTCTGTGCAAGAGGGCCT AATTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA 39 5B7Lightchain GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACA variableregion ACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATG nucleotide GAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAG sequence CGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTT CACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGG AGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCT CAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 40 3D12Heavy GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGTCTTC chainvariable AGTGAGGATATCCTGCAAAGCTTCTGGATACACATTCACTGACTACAACA region TGGACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAACT nucleotide ATTAATCCTAACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAA sequence GGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAACTCC GCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGTGAGA CAGCTCGGGCTGTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTGCA 41 3D12Light GAAATTGTGCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGA chainvariable GAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACT region TGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTAT nucleotide GGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGG sequence ATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATG CTGCCACTTATTACTGTCAACAGTGGAGTAGTTACCCACTCACGTTCGGC TCGGGGACAAAGTTGGAAATAAAA 42 1D2Heavy GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTC chainvariable ACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATA nucleotide GCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGC sequence TACATACACTACAGTGGTAGCACTAACTACAACCCATCTCTCAAAAGTCG AATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGA GTTCTGTGACTACTGAGGACACTGCCACATATTACTGTACAAGAAGCCCG TACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA 43 1D2Lightchain GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGCGA variableregion TCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTGCACAGTGATG nucleotide GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAG sequence CTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTT CAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCG TGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 44 5B7scFV QIQLVQSGPELKKPGETVMISCKASGYSFTNYGMNWVKQAPEKDLKWMGW (linkertwice- INTYTGESRYVDEFKGRFAFSLETSVSIVYLKINNLKNEDMATYFCARGP underlined,any NFDVWGTGTTVTVSSAKTTAPSVYPLAPGSLGGTGGGSGGGGSGGGGSDV suitableknown VMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRL linkermaybe IYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHEPOT used) FGGGTKLEIKRADAAPTVSIFPPSSKLG 45 3D12scFV EVQLQQSGPELVKPGSSVRISCKASGYTFTDYNMDWVKQSHGKSLEWIGT (linkertwice- INPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARVR underlined,any QLGLWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSLGGTGGGSGGGGSGGG suitableknown GSEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPW linkermaybe IYGTSNLASGVPVRESGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLT used) FGSGTKLEIKRADAAPTVSIFPPSSKLG 46 1D2scFV DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMG (linkertwice- YIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYCTRSP underlined,any YFDVWGTGTTVTVSSAKTTPPSVYPLAPGSLGTGGGSGGGGSGGGGSDVV suitableknown MTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLI linkermaybe YKVSNRFSGVPDRESGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTF used) GGGTKLEIKRADAAPTVSIFPPSSKLG 47 Linker GGGSGGGGSGGGGS (exemplary linker,any suitablelinker maybeused) 48 Aminoacidresidues15to37of ACVRACGADSYEMEEDGVRKCKK EGFRvIII,withSer15toAla mutation 49 Aminoacidresidues15to37of SAVRACGADSYEMEEDGVRKCKK EGFRvIII,withCys16toAla mutation 50 Aminoacidresidues15to37of SCARACGADSYEMEEDGVRKCKK EGFRvIII,withVal17toAla mutation 51 Aminoacidresidues15to37of SCVAACGADSYEMEEDGVRKCKK EGFRvIII,withArg18toAla mutation 52 Aminoacidresidues15to37of SCVRAAGADSYEMEEDGVRKCKK EGFRvIII,withCys20toAla mutation 53 Aminoacidresidues15to37of SCVRACAADSYEMEEDGVRKCKK EGFRvIII,withGly21toAla mutation 54 Aminoacidresidues15to37of SCVRACGAASYEMEEDGVRKCKK EGFRvIII,withAsp23toAla mutation 55 Aminoacidresidues15to37of SCVRACGADAYEMEEDGVRKCKK EGFRvIII,withSer24toAla mutation 56 Aminoacidresidues15to37of SCVRACGADSAEMEEDGVRKCKK EGFRvIII,withTyr25toAla mutation 57 Aminoacidresidues15to37of SCVRACGADSYAMEEDGVRKCKK EGFRvIII,withGlu26toAla mutation 58 Aminoacidresidues15to37of SCVRACGADSYEAEEDGVRKCKK EGFRvIII,withMet27toAla mutation 59 Aminoacidresidues15to37of SCVRACGADSYEMAEDGVRKCKK EGFRvIII,withGlu28toAla mutation 60 Aminoacidresidues15to37of SCVRACGADSYEMEADGVRKCKK EGFRvIII,withGlu29toAla mutation 61 Aminoacidresidues15to37of SCVRACGADSYEMEEAGVRKCKK EGFRvIII,withAsp30toAla mutation 62 Aminoacidresidues15to37of SCVRACGADSYEMEEDAVRKCKK EGFRvIII,withGly31toAla mutation 63 Aminoacidresidues15to37of SCVRACGADSYEMEEDGARKCKK EGFRvIII,withVal32toAla mutation 64 Aminoacidresidues15to37of SCVRACGADSYEMEEDGVAKCKK EGFRvIII,withArg33toAla mutation 65 Aminoacidresidues15to37of SCVRACGADSYEMEEDGVRACKK EGFRvIII,withLys34toAla mutation 66 Aminoacidresidues15to37of SCVRACGADSYEMEEDGVRKAKK EGFRvIII,withCys35toAla mutation 67 Aminoacidresidues15to37of SCVRACGADSYEMEEDGVRKCAK EGFRvIII,withLys36toAla mutation 68 Aminoacidresidues15to37of SCVRACGADSYEMEEDGVRKCKA EGFRvIII,withLys37toAla mutation 69 Aminoacidresidues1to76of LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKK EGFRvIII CEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSI SG 70 Aminoacidresidues3to37of EKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKK EGFRvIII 71 Aminoacidresidues1to18of LEEKKGNYVVTDHGSCVR EGFRvill 72 5B7 QIQLVQSGPELKKPGETVMISCKASGYSFTNYGMNWVKQAPEKDLKWMGW ShorterscFv INTYTGESRYVDEFKGRFAFSLETSVSIVYLKINNLKNEDMATYFCARGP (linkertwice- NFDVWGTGTTVTVSSGTGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPA underlined,any SISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTG suitableknown SGSGTDFTLKISRVEAEDLGVYYCWQGTHEPQTFGGGTKLEIK linkermaybe used) 73 5B7scFv QIQLVQSGPELKKPGETVMISCKASGYSFTNYGMNWVKQAPEKDLKWMGW consensus INTYTGESRYVDEFKGRFAFSLETSVSIVYLKINNLKNEDMATYFCARGP (linkertwice- NFDVWGTGTTVTVSS[Linker]DVVMTQTPLTLSVTIGQPASISCKSSQS underlined,any LLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDETL suitableknown KISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEI linkermaybe used) 74 5B7CAR QIQLVQSGPELKKPGETVMISCKASGYSFTNYGMNWVKQAPEKDLKWMGW consensus INTYTGESRYVDEFKGRFAFSLETSVSIVYLKINNLKNEDMATYFCARGP (linkertwice- NFDVWGTGTTVTVSS[Linker]DVVMTQTPLTLSVTIGQPASISCKSSQS underlined,any LLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTL suitableknown KISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIVISNSVMYFSSVVPVLQ linkermaybe KVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFAPSKPF used) WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR KHYQPYAPPRDFAAYRSASLRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 75 3D12 EVQLQQSGPELVKPGSSVRISCKASGYTFTDYNMDWVKQSHGKSLEWIGT ShorterscFV INPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARVR (linkertwice- QLGLWFAYWGQGTLVTVSAGTGGGSGGGGSGGGGSEIVLTQSPALMAASP underlined,any GEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRESG suitableknown SGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGSGTKLEIK linkermaybe used) 76 3D12 EVQLQQSGPELVKPGSSVRISCKASGYTFTDYNMDWVKQSHGKSLEWIGT scFV INPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARVR consensus QLGLWFAYWGQGTLVTVSA[Linker]EIVLTQSPALMAASPGEKVTITCS (linkertwice- VSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRESGSGSGTSYSL underlined,any TISSMEAEDAATYYCQQWSSYPLTFGSGTKLEIK suitableknown linkermaybe used) 77 3D12 EVQLQQSGPELVKPGSSVRISCKASGYTFTDYNMDWVKQSHGKSLEWIGT CAR INPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARVR consensus QLGLWFAYWGQGTLVTVSA[Linker]EIVLTQSPALMAASPGEKVTITCS (linkertwice- VSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRESGSGSGTSYSL underlined,any TISSMEAEDAATYYCQQWSSYPLTFGSGTKLEIKVISNSVMYFSSVVPVL suitableknown QKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFAPSKP linkermaybe FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT used) RKHYQPYAPPRDFAAYRSASLRVKESRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 78 1D2 DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMG ShorterscFV YIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYCTRSP (linkertwice- YFDVWGTGTTVTVSSAGTGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQ underlined,any ASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRES suitableknown GSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK linkermaybe used) 79 1D2 DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMG Consensus YIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYCTRSP scFV(linker YFDVWGTGTTVTVSSA[Linker]DVVMTQTPLSLPVSLGDQASISCRSSQ twice- SLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSGSGTDET underlined,any LKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK suitableknown linkermaybe used) 80 1D2 DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMG CAR YIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYCTRSP Consensus YFDVWGTGTTVTVSSA[Linker]DVVMTQTPLSLPVSLGDQASISCRSSQ (linkertwice- SLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSGSGTDET unerlined,any LKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKVISNSVMYFSSVVPV suitableknown LQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFAPSK linkermaybe PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP used) TRKHYQPYAPPRDFAAYRSASLRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 81 3GGGGS- GGGGSGGGGSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV flexiblelinker HTRGLDFACD (exemplary)and humanCD8 hinge 82 Aminoacid EVQLQQSGPELVKPGSSVRISCKASGYTFTDYNMDWVKQSHGKSLEWIGT sequencefor INPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARVR 3D12bi- QLGLWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSLGGTGGGSGGGGSGGG specificTcell GSEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPW engager IYGTSNLASGVPVRESGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLT exemplary FGSGTKLEIKRADAAPTVSIFPPSSKLGDLGGGGSRDDDIKLQQSGAELA sequence RPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQ Linker KFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQG sequences TTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRA underlined,any SSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTIS suitableknown SMEAEDAATYYCQQWSSNPLTFGAGTKLELK linkermaybe used CD3-specific scFvengager showninbold 83 Linkerofthe PLGGGGSGGGGSGGGGSGGTTTPAPRPPTPAPTIASQPLSLRPEACRPAA L17-CD8 GGAVHTRGLDFACD construct Restrictionsite scarunderlined 84 Linkerofthe PLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 45CD8h construct Restrictionsite scarunderlined 85 Linkerofthe PLPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 35CD8h construct Restrictionsite scarunderlined 86 Linkerofthe PLAGGAVHTRGLDFACD 15CD8h construct Restrictionsite scarunderlined 87 18-merpriorart GGSSRSSSSGGGGSGGGG linkersequence (Andris- Widhopfetal., 2011) 88 G.sub.4Slinker(5- GGGGS mer) 89 (G.sub.4S).sub.3linker GGGGSGGGGSGGGGS (15-mer) 90 (G.sub.4S).sub.4linker GGGGSGGGGSGGGGSGGGGS (20-mer) 91 Human LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIG EGFRvIII IGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQE ectodomain LDILKTVKEITGELLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVV aminoacid SLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKII sequence SNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNL (aminoacids1- LEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHC 354) VKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGP KIPS 92 17AApoly- GGGGSGGGGSGGGGSGG glycinelinker