Nanobody-redirected car t-cells

Abstract

The present disclosure provides mesothelin (MSLN)-specific nanobodies, CEACAM5-specific nanobodies, bispecific MSLN / CEACAM5 antibodies, and chimeric antigen receptors (CARs), including bispecific CARs therefrom. The disclosure further provides CAR T / NK cells and methods of treatment with antibodies and CAR T / NK cells described herein.

Description

[0001] Attorney Docket No: 046483 -7482WO 1(04099)

[0002] NANOBODY-REDIRECTED CAR T-CELLS

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] The present application is entitled to priority under 35 U. S. C. § 119(e) to U. S.

[0005] Provisional Patent Application No. 63 / 736,278 filed on December 19, 2024 and U. S. Provisional Patent Application No. 63 / 736,317 filed on December 19, 2024, each of which are herein incorporated by reference in their entirety.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The XML file named “046483-7482xx.xml” created on December 17, 2025, comprising 258,160 bytes, is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] The development of the chimeric antigen receptor (CAR) and its successful clinical use to direct T cells against specific types of cancers has been an important advancement in cancer immunotherapy. Despite the efficacy of some of these approaches, CAR constructs must be individually designed and developed for each disease, and target molecules must be selected that minimize unintended toxicities to normal cell populations, which also express them. The risk of on-target, off-tumor toxicity can be high, as illustrated by toxicity in Her2-CAR T cell and GD2-CAR T cell clinical trials, for example.

[0010] Thus, there is a need for novel and effective compositions and methods for cancer treatment.

[0011] SUMMARY OF THE INVENTION

[0012] As described herein, the present invention relates to modified immune cells or precursors thereof comprising monospecific nanobodies, bispecific nanobodies, and chimeric antigen receptors (CARs) targeting mesothelin (MSLN) and carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5).

[0013] In one aspect, the present invention provides a nanobody comprising CDR1, CD2, and CDR3 amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; (b) SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, Attorney Docket No: 046483 -7482WO 1(04099)

[0014] respectively; (c) SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; (d) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; (e) SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; (f) SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; (g) SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; (h) SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; and (i) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively, wherein the nanobody comprises an antigen binding domain that specifically binds mesothelin (MSLN) or CEACAM5.

[0015] In an embodiment, the antigen binding domain specifically binds MSLN.

[0016] In another embodiment, the antigen binding domain specifically binds CEACAM5.

[0017] In an embodiment, the nanobody is a VHH comprising the amino acid sequence set forth in any one of SEQ ID NOs: 28-36 and / or encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 37-45.

[0018] In another aspect, the present invention provides a chimeric antigen receptor (CAR), comprising an MSLN antigen binding and / or a CEACAM5 antigen binding domain; a transmembrane domain; and an intracellular domain.

[0019] In an embodiment, the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, and a transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, 0X40 (CD134), 4-1BB (CD137), and CD154. In one embodiment, the transmembrane domain comprises a transmembrane domain of CD8, optionally comprising the amino acid sequence of SEQ ID NO: 51. In another embodiment, the transmembrane domain comprises a transmembrane domain of CD28, optionally comprising the amino acid sequence of SEQ ID NO: 52.

[0020] In an embodiment, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an IgG4 hinge region, an artificial hinge domain, a CD8 hinge region, or any combination thereof. In one embodiment, the hinge domain is an IgG4 hinge, optionally comprising the amino acid sequence of SEQ ID NO: 48.

[0021] In an embodiment, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain. Attorney Docket No: 046483 -7482WO 1(04099)

[0022] In some embodiments, the costimulatory signaling domain comprises a costimulatory signaling domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP 12, CD27, CD2, CD5, ICAM-1, LFA-1, Lek, TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, B7-H3 (CD276), a variant thereof, and a combination thereof. In an embodiment, the costimulatory signaling domain comprises a costimulatory signaling domain of 4-1BB, optionally comprising the amino acid sequence of SEQ ID NO: 55. In another embodiment, the costimulatory signaling domain comprises a costimulatory signaling domain of CD28, optionally comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the CAR comprises two costimulatory signaling domains. In an embodiment, the costimulatory signaling domain comprises a CD28 costimulatory signaling domain and a 4-1BB costimulatory signaling domain, optionally wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 55 and the CD28 costimulatory domain comprises the amino acid sequence of SEQ ID NO: 56.

[0023] In some embodiments, the intracellular signaling domain comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain (CD3, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. In one embodiment, the intracellular signaling domain comprises an intracellular domain of CD3(^, optionally wherein the intracellular domain of CD3(^ comprises the amino acid sequence of SEQ ID NO: 60.

[0024] In an embodiment, the CAR comprises an anti-MSLN nanobody, an IgG4 hinge region, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, and a CD3 intracellular signaling domain.

[0025] In an embodiment, the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 82-89.

[0026] In an embodiment, the CAR comprises an anti-CEACAM5 nanobody, an IgG4 hinge region, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, a 4- IBB costimulatory signaling domain, and a CD3ζ intracellular signaling domain.

[0027] In an embodiment, the CAR comprises the amino acid sequence of SEQ ID NO: 90 or 91. Attorney Docket No: 046483 -7482WO 1(04099)

[0028] In some embodiments, the CAR further comprises a second antigen binding domain. In an embodiment, the second antigen binding domain is an antibody or an antigen-binding fragment thereof. In certain embodiments, the second antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a nanobody. In one embodiment, the second antigen-binding fragment is an scFv. In another embodiment, the second antigen-binding fragment is a nanobody. In certain embodiments, the second antigen binding domain is a tumor-associated antigen (TAA) binding domain. In some embodiment, the TAA binding domain specifically targets a TAA selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA, CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA / CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, variants thereof, and combinations thereof.

[0029] In an embodiment, the CAR comprises an anti-MSLN binding domain and an anti-CEACAM5 binding domain. In an embodiment, the anti-MSLN binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 28-35 and / or encodes the nucleic acid sequence of any one of SEQ ID NO: 37-44, respectively, and the anti-CEACAM5 binding domain comprises the amino acid sequence of SEQ ID NO: 36 and / or encodes the nucleotide sequence of SEQ ID NO: 45.

[0030] In some embodiments, the CAR comprises a self-cleaving 2A peptide domain selected from the group consisting of T2A, P2A, E2A and F2A.

[0031] In certain embodiments, the CAR is a Split Bi-CAR. In one embodiment, the Split BiCAR comprises from its amino to carboxy terminus, the second antigen binding domain, a first costimulatory signaling domain, an intracellular signaling domain, the anti-MSLN nanobody, and a second costimulatory signaling domain. In another embodiment, the Split Bi-CAR comprises from its amino to carboxy terminus, the anti-MSLN nanobody, a first costimulatory signaling domain, an intracellular signaling domain, the second antigen binding domain, and a Attorney Docket No: 046483 -7482WO 1(04099)

[0032] second costimulatory signaling domain. In another embodiment, the Split Bi-CAR comprises from its amino to carboxy terminus, the anti-CEACAM5 nanobody, a first costimulatory domain, an intracellular signaling domain, the second antigen binding domain, and a second costimulatory domain. In another embodiment, the Split Bi-CAR comprises from its amino to carboxy terminus, the second antigen binding domain, a first costimulatory domain, an intracellular signaling domain, the anti-CEACAM5 nanobody, and a second costimulatoiy domain. In another embodiment, the Split Bi-CAR comprises a CD28 costimulatory signaling domain and a 4- IBB costimulatory signaling domain. In another embodiment, the Split Bi-CAR comprises from its amino to carboxy terminus, an anti-CEACAM5 nanobody, a CD28 costimulatory signaling domain, a CD3ζ intracellular signaling domain, the anti-MSLN nanobody, and a 4-1BB costimulatory signaling domain. In another embodiment, the Split Bi-CAR comprises the amino acid sequence of SEQ ID NO: 110 or 111.

[0033] In certain embodiments, the CAR is a Tandem Bi-CAR. In an embodiment, the Tandem Bi-CAR comprises, from its amino to carboxy terminus, the anti-MSLN nanobody, the second antigen binding domain, a first costimulatory signaling domain, a second costimulatory signaling domain, and an intracellular signaling domain. In one embodiment, the Tandem Bi-CAR comprises from its amino to carboxy terminus, the second antigen binding domain, the anti-MSLN nanobody, a first costimulatory signaling domain, a second costimulatory signaling domain, and an intracellular signaling domain. In another embodiment, the Tandem Bi-CAR comprises, from its amino to carboxy terminus, the anti-CEACAM5 nanobody nanobody, the second antigen binding domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain. In another embodiment, the Tandem Bi-CAR comprises, from its amino to carboxy terminus, the second antigen binding domain, the anti-CEACAM5 nanobody nanobody, a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3 intracellular signaling domain. In an embodiment, the Tandem Bi-CAR comprises a CD28 costimulatory signaling domain and a 4- IBB costimulatory signaling domain. In one embodiment, the Tandem Bi-CAR comprises from its amino to carboxy terminus, the anti-CEACAM5 nanobody, an anti-MSLN nanobody, a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, and a CD3(^ intracellular signaling domain. In another embodiment, the Tandem Bi-CAR comprises the amino acid sequence of SEQ ID NO: 114 or

[0034]

[0035] Attorney Docket No: 046483 -7482WO 1(04099)

[0036] In another aspect, the present invention provides a nucleic acid encoding any of the nanobodies or CARs described herein.

[0037] In another aspect, the present invention provides a vector comprising any of the foregoing nucleic acids or others described herein. In some embodiments, the vector is an expression vector. In certain embodiments, the vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In one embodiment, the vector is a lentiviral vector. In certain embodiments, the lentiviral vector is a self-inactivating HIV vector comprising an LTR comprising a U3 deletion. In one embodiment, the HIV vector comprises a partial gag sequence, optionally wherein the partial gag sequence comprises the nucleotide sequence of SEQ ID NO: 133. In another embodiment, the HIV vector further comprises a central polypurine tract (cPPT), optionally wherein the cPPT comprises the nucleotide sequence of SEQ ID NO: 134. In some embodiments, the vector comprises a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE), optionally wherein the WPRE comprises the nucleotide sequence of SEQ ID NO: 135. In some embodiments, the vector comprises the nucleotide sequence of any one of SEQ ID NOs: 124-129.

[0038] In another aspect, the present invention provides a cell comprising the any of the CARs, nucleic acids or vectors described herein. In some embodiments, the cell is an immune cell or precursor cell thereof. In certain embodiments, the immune cell is a T cell or a natural killer (NK) cell. In one embodiment, the T cell is a gamma delta T cell. In some embodiments, the cell is an autologous cell. In other embodiments, the cell is an allogeneic cell. In an embodiment, the cell comprises any of the CARs described herein, any of the nucleic acids described herein, or any of the vectors described herein, wherein the cell expresses a single exogenous antigen binding domain. In another embodiment, the cell comprises any of the CARs described herein, any of the nucleic acids described herein, or any of the vectors described herein, wherein the cell expresses two exogenous antigen binding domains.

[0039] In another aspect, the present invention provides a pharmaceutical composition comprising any of the anti-MSLN and / or anti-CEACAM5 nanobodies described herein, any of the mRNAs encoding an anti-MSLN and / or anti-CEACAM5 nanobody described herein, or any of the cells described herein; and a pharmaceutically acceptable carrier. Attorney Docket No: 046483 -7482WO 1(04099)

[0040] In another aspect, a method for killing a cell comprises contacting the cell with a cell comprising any of the anti-MSLN and / or anti-CEACAM5 nanobodies described herein, any of the CARs described herein, any of the nucleic acids described herein, any of the mRNAs encoding an anti-MSLN and / or anti-CEACAM5 nanobody described herein, or any of the vectors described herein, wherein the cell is an MSLN+and / or CEACAM5+cell. In some embodiments, the cell is a gastric cancer cell, a pancreatic cancer cell, a colorectal cancer cell, a glioma cell, a breast cancer cell, a liver cancer cell, or an ovarian cancer cell.

[0041] In another aspect, a method for treating cancer in a subject in need thereof, comprises administering to the subject an effective amount of any of the pharmaceutical compositions described herein, thereby treating the cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from the group consisting of gastric cancer, pancreatic cancer, colorectal cancer, melanoma, glioma, breast cancer, liver cancer, lung cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, hepatocellular carcinoma, rectal cancer, kidney cancer, prostate cancer, bronchus cancer, urinary bladder cancer, and brain cancer. In some embodiments, the solid tumor is gastric cancer, pancreatic cancer, or colorectal cancer. In some embodiments, the pharmaceutical composition is administered intratumorally. In some embodiments, the cancer is a hematologic cancer. In some embodiments, the hematologic cancer is a myeloid malignancy selected from the group consisting of acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), myelodysplastic neoplasm, and myeloproliferative neoplasm.

[0042] In another aspect, a method for preparing a cell described herein, comprises introducing into the cell any of the nucleic acids or vectors described herein.

[0043] In another aspect, the present invention provides an antibody-drug conjugate, comprising a drug, toxin, or radioisotope conjugated to a nanobody described herein, wherein the nanobody specifically binds MSLN or CEACAM5. In some embodiments, the nanobody specifically binds MSLN. In some embodiments, the nanobody specifically binds CEACAM5. In some embodiments, maytansinoid (DM1), SSTR2-binding octreotide, paclitaxel, auristatin, MMAE, MMAF, dauxrubicin, duocarmycin A, 5-fluoruracil, methotrexate, tubulin polymerization inhibitors, ravtansine (DM4), Ricin A, 90Y, 177Lu, or 11 Un is conjugated to the nanobody. Attorney Docket No: 046483 -7482WO 1(04099)

[0044] In another aspect, the present invention provides a bispecific antibody comprising an anti-MSLN nanobody and an anti-CEACAM5 nanobody as described herein. In some embodiments, the bispecific antibody comprises one or more immunoglobulin constant regions.

[0045] In another aspect, a method for treating cancer in a subject in need thereof, comprises administering to the subject an effective amount of any of the antibody-drug conjugates described herein, thereby treating the cancer.

[0046] In another aspect, a method for treating cancer in a subject in need thereof, comprises administering to the subject an effective amount of any of the bispecific antibodies described herein, thereby treating the cancer.

[0047] In another aspect, a method for treating cancer in a subject in need thereof, comprises administering to the subject an effective amount of any of the anti-MSLN and / or anti-CEACAM5 nanobodies described herein, thereby treating the cancer.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0050] FIGs. 1A-1F: Isolation of anti-CEACAM5 VHHB30 using the STAR system. (A) The flowchart of BON cell-specific CAR-compatible nanobody screening. (B) Confirmation of the isolated VHHB30 binding to BON cells using flow cytometry. (C) Flowchart for screening antigen binding to VHHB30 using HEK293T cells transfected with cDNA plasmids encoding -3,000 human cell surface proteins, followed by flow cytometry with VHH phage and FITC-labeled secondary antibody against phage Ml 3 protein. (D) Flow cytometry analysis of VHHB30 specifically binding to ectopically expressed human CEACAM5, but not murine CEACAM5 in HEK293T cells. (E) Nucleotide sequence of anti-CEACAM5 VHHB30; (F) Amino acid sequence of anti-CEACAM5 VHHB30.

[0051] FIGs. 2A-2D: Identification of the CEACAM5 domain binds to VHHB30. (A) Illustration of the extracellular domains and CEACAM5 truncations, including full-length CEACAM5 and different fragments / deletional mutants of CEACAM5. (B) HEK293T cells were transfected with full-length CEACAM5 (WT) or truncation mutant plasmids, followed by flow analysis with anti-CEACAM5 VHHB30 nanobody. (C) Schematic diagram of VHHB302nd Attorney Docket No: 046483 -7482WO 1(04099)

[0052] and 3rd generation CAR structure, including signal peptide (SP), IgG4 mutant (IgG4m) hinge, CD8 / CD28 transmembrane domain (TM), CD28, 4-1BB, and CD3(^. (D) The expression of CARs in primary human T-cells was detected by flow cytometry with anti-VHH antibody.

[0053] FIGs. 3A-3E: Evaluating the specificity of the VHHB30 CEACAM5 CAR T-cells (CEACAR Ts) in killing of NB4 negative and CEACAM5 -expressing NB4 cells in vitro. (A) Detecting CEACAM5 levels on the surface of NB4 cells using flow assay with anti-CEACAM5 antibody and VHHB30. (B) In vitro cytotoxicity using LDH release assay following overnight co-culture of either control CEACAM5 -negative NB4 cells (left) or CEACAM5-expressing NB4 cells (right) with VHHB30 CEACAR Ts. (C) CEACAM5-expressing NB4 cell specifically induced the IFN-y release of the VHHB30 CEACAR Ts. (D) CEACAM5 -expressing NB4 cell specifically induced the TNF-a release of the VHHB30 CEACAR Ts. (E) CEACAM5-expressing NB4 cell specifically induced the degranulation of the VHHB30 CEACAR Ts.

[0054] FIGs. 4A-4D: VHHB303rd generation CAR T-cells showed a more potent killing effect on CEACAM5-expressing CRC tumor cells. (A) VHHB30 and APC-labelled anti -HA antibody were used as primary and secondary antibodies to detect CEACAM5 expression on various CRC tumor cells, LoVo, HT-29, and SW48. (B) In vitro cytotoxicity using LDH release assay following overnight co-culture of either LoVo, HT-29, and SW-48 with UTD, 2ndand 3rdgeneration VHHB30 CEACAR T-cells. (C-D) ELISA of IFN-y (C) or TNF-a (D) release by VHHB30 CEACAR Ts after overnight co-culture with LoVo, HT-29, and SW-48 tumor cells. Data are presented as the mean ± s.d. (n=3 independent experiments; two-way ANOVA; *P < 0.05, **P < 0.01).

[0055] FIGs. 5A-5G: VHHB30 CEACAR T-cells eliminated CRC tumors in vivo. (A) VHHB30 CEACAR Ts were collected and infused into mice 5 days after virus transduction. (B-D) The antitumor effect of VHHB302nd and 3rd generation CAR T-cells was evaluated in LoVo (B), HT-29 (C), and SW-48 (D) tumor models. Tumor volume was detected twice a week (n=6 tumors / group). E-G. T-cell number in the peripheral blood were measured two weeks after the first administration (n=3 mice / group). Data are presented as mean ± s.d. (two-way ANOVA; *P < 0.05).

[0056] FIGs. 6A-6G: VHHB30 CEACAR T-cells killed GC tumors in vitro and in vivo. (A) Flow cytometry analysis of VHHB30 binding to GC cell lines, SNU-16 and AGS. (B) In vitro cytotoxicity using LDH release assay following overnight co-culture of either AGS or SNU-16 Attorney Docket No: 046483 -7482WO 1(04099)

[0057] with UTD, 2nd and 3rd generation VHHB30 CEACAR T-cells. Data are presented as the mean ± s.d. (n=3 independent experiments; two-way ANOVA; *P < 0.05, **P < 0.01). (C-D) ELISA of IFN-y (C) or TNE-a (D) release by VHHB30 CEACAR T-cells after overnight co-culture with AGS and SNU-16 tumor cells (n=3 independent co-culture). Data are presented as mean ± s.d. (two-tailed unpaired Student’s t-test; **P < 0.01). E. VHHB30 CEACAR T-cells were collected and infused into mice with SNU-16 tumors 5 days after virus transduction. F. Tumor volume was detected twice a week (n=6 tumors / group). Data are presented as mean ± s.d. (two-way ANOVA; **P < 0.01). G. T cell number in the peripheral blood were measured two weeks after the first administration of VHHB30 CEACAR T-cells (n=3 independent co-culture). Data are presented as mean ± s.d. (two-tailed unpaired Student’s t-test; **P < 0.01).

[0058] FIGs. 7A-7J: Specificity analysis of the VHHB30 CEACAR Ts in killing BON and HT-29 tumor xenografts in vivo. (A) Flow cytometry analysis of VHHB30 binding to BON and HT-29 cell lines. (B-C) In vitro cytotoxicity using LDH release assay following overnight co-culture of either BON (B) or HT-29 (C) with UTD and 3rd generation VHHB30 CEACAR T-cells. (D) Flowchart illustrating the treatment of BON and HT-29 xenograft-bearing mice with UTD and VHHB30 CEACAR T-cells. (E, H) Monitoring the growth of BON (E) or HT-29 (H) tumor xenografts treated with either UTD or VHHB30 CEACAR T-cells in vivo (n=6 tumors / group). (F, I) Monitoring the body weight change of mice treated with UTD or VHHB30 CEACAR T-cells in BON (F) or HT-29 (I) tumor xenografts (n=3 mice / group). (G, J) Peripheral blood was collected 14 days post-CART injection to detect the number of CARTs (CD3+VHH+cells) by flow analysis in BON (G) or HT-29 (J) tumor xenografts (n=3 mice / group).

[0059] FIGs. 8A-8C: Generation of anti-MSLN-VHH nanobodies from a llama. (A) A llama was immunized with the purified extracellular domain (296aa-580aa) of human MSLN (13128-H08H1 Sino Biological), and PBMCs were isolated from the immunized llama (Caproligics, MA). RNA was extracted from the PBMCs and used to construct a phage display library using the pComb3x phage display vector. The library was then screened against the MSLN-expressing NB4 cell line. (B) NB4 cells were transduced with recombinant vector expressing human full-length MSLN cDNA (laa-630aa). The cells were then subjected to FACS sorting based on MSLN expression. MSLN-negative and MSLN-expressing NB4 cells were evaluated with flow cytometry assay and Western blot analyses. Of note, during the flow cytometry assay, MSLN-negative and MSLN-expressing NB4 cells were preincubated with the Fc receptor blocker Attorney Docket No: 046483 -7482WO 1(04099)

[0060] (422302, Biolegend) for 15 min, followed by incubation with anti-MSLN antibody (ab!96235, Abeam). (C) DNA sequencing of the isolated anti-MSLN-VHHs identified eight distinct VHHs, i.e. VHH1, 10, 12, 16, 18, 61, 67, 68. Concentrated phages for each of these VHHs were used to bind MSLN-negative or MSLN-expressing NB4 cells, followed by binding to a fluorescence labeled secondary antibody for flow cytometry analysis.

[0061] FIGs. 9A-9C: Amino acid sequences of the isolated MSLN VHHs. (A) The amino acid residue sequence of the MSLN-VHH1, VHH16, VHH68, VHH12, VHH61, VHH10, VHH18, and VHH67 were deduced from Sanger sequencing of the individual phage clones from pComb3x vector. (B) The nucleic acid sequence of each of the unique VHH clones isolated from phages that specifically bound MSLN-expressing NB4 cells, but not MSLN-negative NB4 cells, was obtained from Sanger sequencing of the above VHH clones. (C) The eight MSLN-VHHs and one CEACAM5-VHHB30 nanobodies were expressed and purified from E.coli using Ni-NTA column, and separated in SDS-PAGE, and then transferred to membrane and stained with Ponceau S (top). In separate experiments, the VHH proteins were separated on SDA-PAGE, and then stained with Coomassie blue (bottom), indicating the successful expression of each of the following VHHs, i.e., MSLN-VHH1, VHH16, VHH68, VHH12, VHH61, VHH10, VHH18, and VHH67, and CEACAM5-VHHB30, with CDH17-VHH1 as the control.

[0062] FIGs. 10A-10D: Mapping the domains of MSLN that bind to the generated anti-MSLN-VHHs. (A) Diagram of the precursor protein of MSLN (71 kDa), which is cleaved by Furin into a 40 kDa GPI-anchored, membrane-bound protein, which contains a MUC16 / CA125 binding site and a soluble protein of 31 kDa megakaryocyte-potentiating factor (MPF). (B) Linear illustration of the full-length extracellular part of MSLN comprising MPF, and the three regions of the membrane-anchored part, i.e. RI, RII, and RIII. (C) Protein crystal structure model of MSLN showing RI, RII, and RIII, along with the binding sites of MSLN-VHHs nanobodies. (D) Schema of full-length mature human MSLN and MSLN truncations. HEK293T cells were transfected with plasmids encoding full-length human MSLN or the indicated MSLN truncations or internal deletion, followed by a flow assay with a commercial anti-MSLN antibody [EPR19025-42] (Ab 196235, Abeam) targeted MSLN-RII or a commercial anti-MSLN antibody [5R9H8] (MAS-42914, Invitrogen) targeted MSLN-RIII or one of the eight anti-MSLN-VHH nanobodies.

[0063] Experiments were independently performed three times with similar results. Attorney Docket No: 046483 -7482WO 1(04099)

[0064] FIGs. 11 A-l 1J: Generation of MSLN-VHH CARTs and evaluation of specificity in killing MSLN-expressing NB4 cells in vitro. (A) Diagram illustrating the 3rd generation MSLN-VHHs redirected CARs, including signal peptide (SP), IgG4 mutant (IgG4m) hinge, CD28 transmembrane domain (TM), 2 stimulatory domains (CD28, 4- IBB), and 1 activation domain (CD3Q. (B) Evaluation of VHH expression on the primary total T-cells transduced with the indicated MSLN-VHH CARs lentivirus using flow cytometry assay with anti-VHH antibody. (C) Flow cytometry analysis demonstrating the binding of single MSLN-VHHs CARTs to human MSLN using the anti-human MSLN-His antibody and anti-His secondary antibody. (D) Flow cytometry analysis of the binding of MSLN-VHH single CARTs to mouse MSLN using the antimouse MSLN-His antibody and anti-His secondary antibody. (E-F). In vitro cytotoxicity using LDH release assay following overnight co-culture of either control MSLN-negative NB4 cells (E) or MSLN-expressing NB4 cells (F) with each of the eight MSLN-VHH CARTs (n=3 repeats). (G-H) ELISA assay for detecting TNF-a cytokine release from UTD control T-cells or each of the MSLN-VHH CARTs in co-culture with the MSLN-negative NB4 (G) or MSLN-expressing NB4 cells (H) (n=3 repeats). (I -J) ELISA assay for detecting IFN-y cytokine release from UTD control T-cells or each of the MSLN-CARTs in co-culture with the MSLN-negative NB4 (I) or MSLN-expressing NB4 cells (J) (n=3 repeats).

[0065] FIGs. 12A-12C: Ectopic expression of human MSLN in NB4 cells did not affect the cell growth and differential ability of the single MSLN-VHH CARTs to suppress ASPC1 tumor xenografts. (A) MSLN-negative NB4 cells and MSLN-expressing NB4 cells were seeded at a density of 0.1 x 106 / mL, and counted on the indicated days: triplicate were used. (B) Growth of ASPC1 tumor xenografts from NSG mice treated with various single MSLN-CARTs was monitored. The data were plotted at an enlarged scale to further compare the different antitumor activity among the various single MSLN-CARTs. (n=6 tumors / group). Data are presented as mean ± s.d. (C) Statistical comparison of antitumor activity in ASPC1 tumor xenografts for VHH1.28BBz and VHH12.28BBz MSLN CARTs against other VHH MSLN CARTs. Statistical comparisons were performed using one-way univariate ANOVA analysis, ****p<0.0001.

[0066] FIG. 13A-13G: Specificity analysis of the MSLN-VHH CAR T-cells in killing MSLN-expressing NB4 tumor xenografts in vivo. (A) Flowchart illustrating the establishment of MSLN-negative or MSLN-expressing NB4 tumor xenografts in NSG mice. (B-C) The mice were then injected via tail vein with either UTD T-cells or one of the eight MSLN-VHH CARTs. The Attorney Docket No: 046483 -7482WO 1(04099)

[0067] growth of MSLN-negative NB4 tumor xenografts (B) or MSLN-expressing NB4 tumor xenografts (C) was monitored (n=6 tumor s / group). (D-E) PBMCs was collected 18 days post-CART injection to detect the number of CARTs (CD3+VHH+cells) by flow analysis in MSLN-negative NB4 tumor xenografts (D) or MSLN-expressing NB4 tumor xenografts (E) (n=3 mice / group). (F-G) Monitoring the body weight change of mice treated with UTD control T-cells or the single MSLN-CARTs in MSLN-negative NB4 tumor xenografts (F) or MSLN-expressing NB4 tumor xenografts (G) (n=3 mice / group). Data are presented as mean ± s.d. Statistical comparisons were performed using one-way univariate ANOVA analysis, ****p<0.0001.

[0068] FIGs. 14A-14H: Evaluating the antitumor activity of the MSLN-VHH CAR T-cells in low MSLN-expressing GI tumor xenografts. (A) MSLN level on the surface of NCI-N87 (Gastric), ASPC1 (Pancreatic), SW48 (Colorectal), and LoVo (Colorectal) cells detected by flow assay with an anti-MSLN antibody (Abl96235, Abeam). (B) MSLN level on NCI-N87, ASPC1, SW48, and LoVo tumor xenografts assessed by IHC staining with an anti-MSLN antibody (Ab 196235, Abeam). Scale bars, 50 pM. (C) Flowchart illustrating the treatment of various MSLN-expressing NCI-N87, ASPC1, SW48, and LoVo xenograft-bearing mice with UTD T-cells or each of the single MSLN-VHH CARTs. (D-E) Monitoring the growth of NCI-N87 tumor xenografts (D) or ASPC 1 tumor xenografts (E) treated with either UTD or the MSLN-VHH CARTs in vivo (n=6 tumors / group). (F-G) Evaluating the growth of SW48 tumor xenografts (F) or low MSLN-expressing LoVo tumor xenografts (G) treated with either UTD or the MSLN-VHH CARTs in vivo (n=6 tumors / group). Mice sacrificed when the tumor diameter exceeds 2 cm3. Data are presented as mean ± s.d. Statistical comparisons were performed using one-way univariate ANOVA analysis, ****p<0.0001.

[0069] FIGs. 15A-15H: Evaluation of peripheral blood CAR T-cell number and body weight change in Gl-tumor xenograft-bearing mice treated with UTD or single MSLN-VHH CAR T-cells. (A) Number of CARTs (CD3+VHH+cells) in peripheral blood of mice bearing NCI-N87 tumor xenografts was detected on the 18th day after CART injection by flow assay (n=3 mice / group). (B) Body weight of UTD or C ARTs-treated mice bearing NCI-N87 tumor xenografts on the indicated days (n=3 mice / group). (C) Number of CARTs (CD3+VHH+cells) in peripheral blood of mice bearing ASPC1 tumor xenografts examined on the 18th day following the CART injection using flow assay (n=3 mice). (D) Body weight of UTD or the single MSLN-CARTs-treated mice bearing ASPC1 tumor xenografts (n=3 mice / group. (E). Number of CARTs Attorney Docket No: 046483 -7482WO 1(04099)

[0070] (CD3+VHH+cells) in peripheral blood of mice bearing SW48 tumor xenografts was tested on the 18th day after CART injection by flow assay (n=3 mice). (F) Body weight of UTD, single MSLN-CARTs-treated mice bearing low MSLN expression SW48 tumor xenografts (n=3 mice / group). (G) Number of CARTs (CD3+VHH+cells) in peripheral blood of mice bearing LoVo tumor xenografts was tested on the 18th day after CART injection by FACS (n=3 mice / group). (H) Body weight of UTD, single MSLN-CAR T-cell -treated mice bearing low MSLN expression LoVo tumor xenografts (n=3 mice per group). Data are presented as the mean ± s.d.

[0071] FIGs. 16A-16E: Generation of bispecific MSLN / CEACAM5 CAR T-cells, i.e. split bispecific CARs (sbiCARs) and tandem bispecific CARs (tbiCARs). (A) Diagram illustrating split CEA / MSLN CAR constructs with two distinct MSLN-VHH binders tethered to different intracellular signaling domains (left vs right). (B) Diagram illustrating tandem CEACAM5 / MSLN CAR constructs with both distinct TAA binders arranged in different orders in a head-tail configuration, both tethered to the same intracellular signaling domains. Note that the sequence / order of the two binders is different. (C-E) Flow cytometry was performed to examine CAR expression on the surface of human primary T-cells transduced with single VHHCARs, sbiCARs, or tbiCARs, using anti-VHH antibody (C), anti-human MSLN-His antibody (D), or anti-human CEACAM5-His antibody (E).

[0072] FIGs. 17A-17G: MSLN / CEACAM5 sbiCAR T-cells potently suppress CEACAM5-expressing / MSLN -negative BON (PanNET) tumor xenografts. (A) Flow assay for detecting MSLN and CEACAM5 levels on the surface of BON cells with anti-MSLN antibody (Abl96235, Abeam), anti-CEACAM5 antibody (ab!33633, Abeam), followed by anti-rabbit IgG antibody (406414, Biolegend). (B) IHC staining of BON tumor xenografts for MSLN (left) or CEACAM5 (right) levels using either anti-MSLN or anti-CEACAM5 antibodies. Scale bars, 50 pM. (C) Flowchart illustrating the treatment of MSLN-negative, CEACAM5-expressing BON xenografts, or dual MSLN / CEACAM5-expressing NCLN87 xenografts with UTD T-cells, single MSLN CAR T cells, single CEACAM5-CAR T-cells, or various bispecific MSLN / CEACAM5 CAR T-cells. (D) Monitoring the impact of the single or MSLN / CEACAM5 biCARTs on the growth of MSLN-negative, CEACAM5 -expressing BON tumor xenografts (n=6 tumors / group). (E) Body weight of UTD control T-cells, or single or MSLN / CEACAM5 biCAR T-cell-treated mice bearing MSLN-negative, CEACAM5 -expressing BON tumor xenografts (n=3 mice / group). Attorney Docket No: 046483 -7482WO 1(04099)

[0073] (F) Monitoring the impact of single or MSLN / CEACAM5 biCARTs on the growth of high MSLN-expressing, and high CEACAM5-expressing NCI-N87 tumor xenografts (n=6 tumors / group). (G) Body weight of UTD, single or bispecific MSLN / CEACAM5 CAR T-cell-treated mice bearing theNCI-N87 tumor xenografts (n=3 mice / group). Data are presented as mean ± s.d. Statistical comparison were performed using one-way univariate ANOVA analysis, ****p<0.0001.

[0074] FIGs. 18A-18I: Evaluation of the efficacy of the bispecific MSLN / CEACAM5 CARTs in killing low MSLN-expressing and low CEACAM5-expression SW48 and LoVo xenografts in vivo. (A) CEACAM5 levels on the surface of NCI-N87 (Gastric), ASPC1 (Pancreatic), SW48 (Colorectal), and LoVo (Colorectal) cells was evaluated with the flow assay. (B) CEACAM5 levels in NCI-N87, ASPC1, SW48, and LoVo tumor xenografts were detected by IHC staining with an anti-CEACAM5 antibody. Scale bars, 50 pM. (C) Flowchart for treating low MSLN-expressing, low CEACAM5 -expressing SW48, and LoVo xenografts with UTD T-cells, single MSLN- or CEACAM5 -CARTs, or various bispecific MSLN / CEACAM5 CARTs. (D) Comparison of antitumor activity between MSLN / CEACAM5 sbiCARTs and single MSLN or CEACAM5-CAR-Ts in suppressing low MSLN-expressing SW48 tumor xenografts (n=6 tumors / group). (E) Comparison of antitumor activity between tandem MSLN / CEACAM5 tbiCARTs and single MSLN- or CEACAM5-CARTs on suppressing low MSLN-expressing SW48 tumor xenografts (n=6 tumors / group). The experiments were performed as in D, but plotted differently to highlight the effect of tbiCARs. (F) Comparison of the antitumor activity between MSLN / CEACAM5 sbiCARTs and single MSLN / CEACAM5 CARTs on suppressing low MSLN-expressing, but CEACAM5-expressing LoVo tumor xenografts (n=6 tumors / group). (G) Comparison of the antitumor activity between tandem MSLN / CEACAM5 tbiCARTs and single MSLN- or CEACAM5-CARTs on suppressing very low MSLN-expressing, but CEACAM5-expressing LoVo tumor xenografts (n=6 tumors / group). (H) Comparison of antitumor activity between MSLN / CEACAM5 sbiCARTs and tbiCARTs in suppressing low MSLN-expressing SW48 tumor xenografts (n=6 tumors / group), VHHB30.28z / VHH12-BB vs VHHB30.28z / VHHl-BB vs. the other sbiCARs and tbiCARTs. (I) Comparison of antitumor activity between MSLN / CEACAM5 sbiCARTs and tbiCARTs in suppressing low MSLN-expressing LoVo tumor xenografts (n=6 tumors / group). The experiments were performed as in (D) but plotted differently to highlight the effect of tbiCARs. Data are presented as mean ± s.d. Attorney Docket No: 046483 -7482WO 1(04099)

[0075] Statistical comparisons were performed using one-way univariate ANOVA analysis, ****p<0.0001.

[0076] FIGs. 19A-19B: Construction of MSLN / CEACAM5 sbiCARs and tbiCARs, revealing two sbiCARs as the most potent CARs suppressing low TAA-expressing tumor xenografts. (A) Schema of split CEA / MSLN CAR constructs with the specific restriction enzymes for the cloning sites. (B) Schema of tandem CEA / MSLN CAR constructs.

[0077] FIGs. 20A-20F: Comparison of VHHB30 / VHH1 biCAR and VHHB30 / VHH1 biCAR with other biCARs. (A-B) Statistical comparisons of antitumor activity of HHB30.28z / VHHl. BB or VHH12. BB sbiCARTs with other biCARTs in SW48 (A) or LoVo (B) bearing mice. (C) Body weight of UTD, single, or bispecific MSLN / CEACAM5 CARTs-treated mice bearing SW48 tumor xenografts (n=3 mice / group). (D) Body weight of UTD or bispecific MSLN / CEACAM5 CARTs-treated mice bearing LoVo tumor xenografts (n=3 mice / group). (E-F) Number of CART cells (CD3+VHH+cells) in the peripheral blood of mice bearing SW48 (E) tumor xenografts (n=3 mice) or LoVo (F) tumor xenografts (n=3 mice) were measured on the 18th day following injections with UTD, single CART, or bispecific MSLN / CEACAM5 by flow assay.

[0078] DETAILED DESCRIPTION

[0079] The present invention provides compositions and methods of using genetically modified immune cells or precursors thereof (e.g., modified T cells) comprising engineered monospecific and bispecific chimeric antigen receptors (CARs) specific for mesothelin (MSLN) or MSLN / CEACAM5.

[0080] Definitions

[0081] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting. Attorney Docket No: 046483 -7482WO 1(04099)

[0082] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art, or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0083] That the disclosure may be more readily understood, select terms are defined below. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0084] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0085] “Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0086] As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0087] The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Attorney Docket No: 046483 -7482WO 1(04099)

[0088] Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell, or a biological fluid.

[0089] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0090] A “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.

[0091] A “co-stimulatory signal,” as used herein, refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0092] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0093] The term “downregulation” as used herein refers to the decrease or elimination of gene expression of one or more genes.

[0094] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or Attorney Docket No: 046483 -7482WO 1(04099)

[0095] prophylactic benefit. Such results may include, but are not limited to an amount that when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0096] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0097] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue, or system.

[0098] The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and / or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and / or sugars in size. Preferably, the epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope. Attorney Docket No: 046483 -7482WO 1(04099)

[0099] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0100] The term “expand” as used herein refers to increasing in number, as in an increase in the number of T cells. In one embodiment, the T cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, the T cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term "ex vivo" as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0101] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0102] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0103] As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0104] As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

[0105] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are Attorney Docket No: 046483 -7482WO 1(04099)

[0106] identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0107] The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0108] The term “immunosuppressive” is used herein to refer to reducing overall immune response.

[0109] “Insertion / deletion,” commonly abbreviated “indel,” is a type of genetic polymorphism in which a specific nucleotide sequence is present (insertion) or absent (deletion) in a genome.

[0110] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0111] The term “knockout” as used herein refers to the ablation of gene expression of one or more genes from a cell.

[0112] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0113] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids. Attorney Docket No: 046483 -7482WO 1(04099)

[0114] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0115] The terms “nanobody,” “VHH domain,” “VHH,” and “VHH nanobody” are used interchangeably with reference to a single variable binding domain of a heavy chain antibody from a camelid animal, such as a camel, a llama, an alpaca, or from a nurse shark.

[0116] The term “VHH domain” has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VH domains” or “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains” or “VL domains”). VHH domains can specifically bind to an epitope without an additional antigen binding domain (as opposed to VH or VL domains in a conventional 4-chain antibody, in which case the epitope is recognized by a VL domain together with a VH domain). VHH domains are small, robust and efficient antigen recognition units formed by a single immunoglobulin domain.

[0117] The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T.”

[0118] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0119] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0120] The term “polynucleotide” as used herein is defined as a chain of nucleotides.

[0121] Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic Attorney Docket No: 046483 -7482WO 1(04099)

[0122] acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0123] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0124] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen.

[0125] However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an Attorney Docket No: 046483 -7482WO 1(04099)

[0126] antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0127] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as upregulation of interferon-gamma, and / or reorganization of cytoskeletal structures, and the like.

[0128] A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.

[0129] A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

[0130] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

[0131] A “target site” or “target sequence” refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

[0132] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (0) chain, although in some cells the TCR consists of gamma and delta (y / 8) chains. TCRs may exist in alpha / beta and Attorney Docket No: 046483 -7482WO 1(04099)

[0133] gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.

[0134] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0135] “Transplant” refers to a biocompatible lattice or a donor tissue, organ, or cell, to be transplanted. An example of a transplant may include but is not limited to skin cells or tissue, bone marrow, and solid organs such as heart, pancreas, kidney, lung, and liver. A transplant can also refer to any material that is to be administered to a host. For example, a transplant can refer to a nucleic acid or a protein.

[0136] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0137] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0138] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0139] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of Attorney Docket No: 046483 -7482WO 1(04099)

[0140] the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0141]

[0142] As further described below, the present invention provides mesothelin (MSLN)-specific and CEACAM5-specific binding domains, MSLN-specific and CEACAM5-specific chimeric antigen receptors (CARs; e.g., an “anti -MSLN CAR”, “MSLN CAR”, “anti-CEACAM5 CAR”, “CEACAM5 CAR”), bi specific CARs, and modified cells comprising the same. Also provided are compositions and methods for utilizing monospecific MSLN and CEACAM5 CARs and bispecific MSLN / CEACAM5 CARs to kill or eliminate cancer cells and to treat MSLN+and / or CEACAM5+cancers and other MSLN+and / or CEACAM5+associated diseases or disorders.

[0143] Chimeric Antigen Receptor (CAR)

[0144] The present invention provides compositions and methods for modified immune cells or precursor cells thereof, e.g., modified T cells, comprising a chimeric antigen receptor (CAR) having specific binding affinity for mesothelin (MSLN), CEACAM5, or both.

[0145] In one aspect, CAR of the present invention comprises an MSLN antigen binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

[0146] In another aspect, the CAR of the present invention comprises a CEACAM5 antigen binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

[0147] The MSLN CAR or CEACAM5 CAR may further comprise a hinge domain (or region). In some embodiments, each of the domains of the MSLN CAR or CEACAM5 CAR is separated by a linker or spacer.

[0148] MSLN Antigen Binding Domain Attorney Docket No: 046483 -7482WO 1(04099)

[0149] In an embodiment, the MSLN antigen binding domain of the invention comprises a nanobody (VHH or single domain antibody) that specifically binds to an MSLN molecule on a cell surface and has specific binding affinity for MSLN+cancer cells. The MSLN binding domain comprises a variable heavy chain (VHH) domain comprising CDR1, CDR2, and CDR3 binding regions. In an embodiment, the MSLN binding domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. In another embodiment, the MSLN binding domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the MSLN binding domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In another embodiment, the MSLN binding domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In another embodiment, the MSLN binding domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15. In another embodiment, the MSLN binding domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 18. In another embodiment, the MSLN binding domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 21. In another embodiment, the MSLN binding domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 24.

[0150] In an embodiment, the MSLN nanobody comprises a VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 28-35. In an embodiment, the MSLN nanobody comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 28. In another embodiment, the MSLN nanobody comprises a VHH domain comprising the amino acid Attorney Docket No: 046483 -7482WO 1(04099)

[0151] sequence of SEQ ID NO: 29. In another embodiment, the MSLN nanobody comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 30. In another embodiment, the MSLN nanobody comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 31. In another embodiment, the MSLN nanobody comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 32. In another embodiment, the MSLN nanobody comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 33. In another embodiment, the MSLN nanobody comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 34. In another embodiment, the MSLN nanobody comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 35.

[0152] In another embodiment, the MSLN nanobody comprises a VHH domain encoded by the nucleotide sequence of any one of SEQ ID NOs: 37-44. In one embodiment, the MSLN nanobody comprises a VHH domain encoded by the nucleotide sequence of SEQ ID NO: 37. In another embodiment, the MSLN nanobody comprises a VHH domain encoded by the nucleotide sequence of SEQ ID NO: 38. In another embodiment, the MSLN nanobody comprises a VHH domain encoded by the nucleotide sequence of SEQ ID NO: 39. In another embodiment, the MSLN nanobody comprises a VHH domain encoded by the nucleotide sequence of SEQ ID NO: 40. In another embodiment, the MSLN nanobody comprises a VHH domain encoded by the nucleotide sequence of SEQ ID NO: 41. In another embodiment, the MSLN nanobody comprises a VHH domain encoded by the nucleotide sequence of SEQ ID NO: 42. In another embodiment, the MSLN nanobody comprises a VHH domain encoded by the nucleotide sequence of SEQ ID NO: 43. In another embodiment, the MSLN nanobody comprises a VHH domain encoded by the nucleotide sequence of SEQ ID NO: 44.

[0153] In some embodiments, the MSLN binding domain comprises a VHH domain comprising an amino acid sequence having at least 95%-99% identity to the amino acid sequence of any one of SEQ ID NOs: 28-35. In some embodiments, the MSLN binding domain comprises a VHH domain encoded by a nucleotide sequence having at least 95%-99% identity to the nucleotide sequence of any one of SEQ ID NOs: 37-44. In some embodiments, the MSLN binding domain is a humanized MSLN binding domain.

[0154] CEACAM5 Antigen Binding Domain

[0155] In an embodiment, the CEACAM5 antigen binding domain of the invention comprises a nanobody (VHH or single domain antibody) that specifically binds to a CEACAM5 molecule on Attorney Docket No: 046483 -7482WO 1(04099)

[0156] a cell surface and has specific binding affinity for CEACAM5+cancer cells. The CEACAM5 binding domain comprises a variable heavy chain (VHH) domain comprising CDR1, CDR2, and CDR3 binding regions. In an embodiment, the CEACAM5 binding domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 27. In an embodiment, the CEACAM5 nanobody comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 36. In another embodiment, the CEACAM5 nanobody comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 45, or 46. In some embodiments, the antigen binding domain comprises a heavy chain variable (VH) region comprising an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 36 and / or a nucleotide sequence having at least 95-99% identity to the nucleotide sequence of SEQ ID NO: 45 or 46. In some embodiments, the CEACAM5 binding domain is a humanized CEACAM5 binding domain.

[0157] Bispecific CARs

[0158] In some embodiments, a bispecific CAR of the present invention comprises a second antigen binding domain which may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a nonhuman antibody, and any fragment thereof. Thus, in one embodiment, the antigen binding domain portion comprises a mammalian antibody or a fragment thereof. In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), and a single-chain variable fragment (scFv).

[0159] In some embodiments, the second antigen binding domain targets a second tumor-associated antigen (TAA). In some embodiments, the tumor associated antigen is overexpressed in a solid tumor. Exemplary tumor associated antigens include, but are not limited to, alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA, CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA / CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D- Attorney Docket No: 046483 -7482WO 1(04099)

[0160] Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, Pl 6, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any variants or combination thereof.

[0161] In certain embodiments, the additional antigen binding domain targets a tumor associated antigen, which is a B cell antigen expressed in a non-solid tumor. Exemplary B cell antigens include but are not limited to CD5, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD33 (IL3Ra), CD34, CD37, CD38, CD40, CD52, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269. Flt3, ROR1, BCMA, FcRn5, FcRn2, CS-L CXCR4, 5, 7, IL-7 / 3R, IL7 / 4 / 3R, and L4R, and any variants or combinations thereof.

[0162] In certain embodiments the second antigen binding domain is a single-chain variable fragment or scFv. As used herein, the terms “single-chain variable fragment” and “scFv” refer to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH: VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker or spacer, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The terms “linker” and “spacer” are used interchangeably herein. In some embodiments, the second antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VH - linker - VL. In some embodiments, the second antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL - linker - VH. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.

[0163] The linker is typically rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers such as (GS)n, (GSGGS)n (SEQ ID NO: 137), (GGGS)n(SEQ ID NO: 138), and (GGGGS)n(SEQ ID NO: 139), where n represents an integer of at least 1. Exemplary linker sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 140), GGSGG (SEQ ID NO: 141), GSGSG (SEQ ID NO: 142), GSGGG (SEQ ID NO: 143), GGGSG (SEQ ID NO: 144), GSSSG Attorney Docket No: 046483 -7482WO 1(04099)

[0164] (SEQ ID NO: 145), GGGGS (SEQ ID NO: 146), (GGGGS)3(SEQ ID NO: 147), (GGGGS)4(SEQ ID NO: 148), and the like. Those of skill in the art would be able to select the appropriate linker sequence for use in the present invention. In some embodiments, an antigen binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL is separated by the linker sequence having the amino acid sequence (GGGGS)3(SEQ ID NO: 147) or (GGGGS)4(SEQ ID NO: 148).

[0165] Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U. S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U. S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g, Zhao et al., Hybridoma (Larchmt) 200827(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 200797(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 19973(3):173-84; Moosmayer et al., Ther Immunol 19952(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g, Peter et al., J Biol Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., CritRev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0166] Tolerable variations of the antigen binding domain will be known to those of skill in the art, while maintaining specific binding to MSLN or CEACAM5. For example, in some embodiments the MSLN binding domain comprises an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the corresponding amino acid sequences set forth in SEQ ID NOs: 28-35 and / or their corresponding nucleotide sequences set forth in SEQ ID NOs: 37-44, respectively. In other embodiments, the CEACAM5 binding domain comprises an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36 and / or the nucleotide sequence set forth in SEQ ID NO: 45 or 46. Attorney Docket No: 046483 -7482WO 1(04099)

[0167] The antigen binding domain may be operably linked to another domain of the CAR, such as a signal peptide domain, hinge region, and / or transmembrane domain as further described below. In one embodiment, a nucleic acid encoding the antigen binding domain is operably linked to a nucleic acid encoding a signal peptide domain and a nucleic acid encoding a hinge region or transmembrane domain.

[0168] The antigen binding domains described herein, such as the antibody or fragment thereof, can be combined with any of the signal peptide domains, hinge regions, transmembrane domains costimulatory signaling domains, or intracellular signaling domains described herein, unless specifically described to the contrary or clearly evident to one skilled in the art.

[0169] Transmembrane Domain

[0170] With respect to the transmembrane domain, the MSLN CAR or CEACAM5 CAR of the present invention comprises a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain. The transmembrane domain is a region that is capable of spanning the plasma membrane of a cell (.g., an immune cell or precursor thereof). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of the MSLN or CEACAM5 CAR. In some embodiments, the transmembrane domain is interposed between a hinge region and the intracellular domain of the MSLN or CEACAM5 CAR.

[0171] In an embodiment, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0172] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane regions of particular use in this invention include, without limitation, transmembrane domains derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain Attorney Docket No: 046483 -7482WO 1(04099)

[0173] of the T-cell receptor, CD28, CD2, CD3 epsilon, MSLN, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In certain exemplary embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0174] In some embodiments, the transmembrane domain comprises a CD8ot transmembrane domain. In one embodiment, the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0175] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 52.

[0176] In some embodiments, the transmembrane domain comprises a ICOS transmembrane domain. In one embodiment, the ICOS transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 53.

[0177] In some embodiments, the transmembrane domain comprises an 0X40 transmembrane domain. In one embodiment, the 0X40 CD28 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 54.

[0178] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains described herein, any of the intracellular signaling domains described herein, or any of the other domains described herein that may be included in the MSLN or CEACAM5 CAR, unless specifically described to the contrary or clearly evident to one skilled in the art.

[0179] In some embodiments, the MSLN CAR or CEACAM5 CAR further includes a hinge region. The hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a Attorney Docket No: 046483 -7482WO 1(04099)

[0180] CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4).

[0181] In some embodiments, the MSLN CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain. The hinge region is capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g, Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). Generally, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell. The flexibility of the hinge region permits the hinge region to adopt many different conformations.

[0182] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0183] The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, or from about 40 amino acids to about 50 amino acids.

[0184] Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g, Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0185] For example, hinge regions can include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 137), (GGGS)n (SEQ ID NO: 138), and (GGGGS)n(SEQ ID NO: 139), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g, Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge Attorney Docket No: 046483 -7482WO 1(04099)

[0186] regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 140), GGSGG (SEQ ID NO: 141), GSGSG (SEQ ID NO: 142), GSGGG (SEQ ID NO: 143), GGGSG (SEQ ID NO: 144), GSSSG (SEQ ID NO: 145), GGGGS (SEQ ID NO: 146), (G4S)3(SEQ ID NO: 147), (G4S)4(SEQ ID NO: 148), and the like.

[0187] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g, Tan et al., Proc. Natl. Acad. Set. USA (1990) 87(1): 162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 149); CPPC (SEQ ID NO: 150); CPEPKSCDTPPPCPR (SEQ ID NO: 151) (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO: 152); KSCDKTHTCP (SEQ ID NO: 153); KCCVDCP (SEQ ID NO: 154); KYGPPCP (SEQ ID NO: 155); EPKSCDKTHTCPPCP (SEQ ID NO: 156) (human IgGl hinge); and the like.

[0188] The hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge region can include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally-occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 156); see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. Exemplary IgG hinge regions include ERKCCVECPPCP (SEQ ID NO: 157) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 158) (human IgG3 hinge); and SPNMVPHAHHAQ (SEQ ID NO: 159) (human IgG4 hinge).

[0189] In one embodiment, the CAR comprises a human IgG4 hinge region comprising the amino acid sequence of SEQ ID NO: 48. In another embodiment, the CAR comprises a human CD8 hinge region comprising the amino acid sequence of SEQ ID NO: 49. In another embodiment, the CAR comprises a human CD28 hinge region comprising the amino acid sequence of SEQ ID NO: 50.

[0190] The transmembrane domain may be combined with any hinge domain and / or may comprise one or more transmembrane domains described herein. For example, in one embodiment, the CAR comprises an IgG4 hinge region of SEQ ID NO: 48 and a CD28 transmembrane domain of SEQ ID NO: 52. Attorney Docket No: 046483 -7482WO 1(04099)

[0191] The transmembrane domains described herein, such as a transmembrane region of alpha, beta or zeta chain of the T-cell receptor, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9, can be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains or intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in the CAR.

[0192] In one embodiment, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In exemplary embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0193] Tolerable variations of the transmembrane and / or hinge domain will be known to those of skill in the art, while maintaining its intended function. For example, in some embodiments a transmembrane domain or hinge domain comprises an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to their corresponding domains from where they are derived or with respect to the sequences in Table 1 herein.

[0194] Intracellular Domain

[0195] The MSLN and CEACAM5 CARs of the present invention also includes an intracellular domain. The intracellular domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed e.g., immune cell). The intracellular domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and / or destroying a target cell.

[0196] The intracellular domain or otherwise the cytoplasmic domain of the CAR is responsible for activation of the cell in which the CAR is expressed. Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability. Attorney Docket No: 046483 -7482WO 1(04099)

[0197] In certain embodiments, the intracellular domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular domain comprises an intracellular signaling domain. In certain embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

[0198] In certain embodiments, the intracellular domain comprises a costimulatory signaling domain comprising any suitable portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8, CD27, CD28, 0X40, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, or any combination thereof.

[0199] In some embodiments, the costimulatory signaling domain is derived from 4-1BB, CD28, ICOS, or 0X40. In an embodiment, the costimulatory signaling domain is derived from 4- IBB and / or comprises an amino acid sequence set forth in SEQ ID NO: 55. In another embodiment, the costimulatory signaling domain is derived from CD28 and / or comprises an amino acid sequence set forth in SEQ ID NO: 56. In another embodiment, the costimulatory signaling domain is derived from ICOS and / or comprises an amino acid sequence set forth in SEQ ID NO: 57. In another embodiment, the costimulatory signaling domain is derived from 0X40 and / or comprises an amino acid sequence set forth in SEQ ID NO: 58.

[0200] Intracellular signaling domains suitable for use in the CARs of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e.g, one, two, three, four, five, six, etc.) IT AM motif as described below. In some embodiments, the intracellular signaling domain includes DAP10 / CD28 type signaling chains. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm.

[0201] Intracellular signaling domains suitable for use in the CARs of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, an IT AM motif is repeated twice in an Attorney Docket No: 046483 -7482WO 1(04099)

[0202] intracellular signaling domain, where the first and second instances of the IT AM motif are separated from one another by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain in the MSLN CAR comprises 3 IT AM motifs. In some embodiments, intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine-based activation motifs (IT AMs) such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. (2014) Immunol. 5:254).

[0203] A suitable intracellular signaling domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0204] Examples of the intracellular signaling domain include, without limitation, the, chain of the T cell receptor complex or any of its homologs, e.g., r| chain, FcsRIy and 0 chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (A, 8 and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.

[0205] Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but are not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma Rl la, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, 0X40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, Attorney Docket No: 046483 -7482WO 1(04099)

[0206] CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD lid, ITGAE, CD 103, ITGAL, CDlla, LFA-1, ITGAM, CD lib, ITGAX, CDllc, 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, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, or any combination thereof.

[0207] Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0208] While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0209] In one embodiment, the intracellular signaling domain in the CAR includes the cytoplasmic signaling domain of human CD3 zeta. In an exemplary embodiment, the intracellular signaling domain of human CD3 zeta comprises the amino acid sequence set forth in SEQ ID NO: 59 or 60. Attorney Docket No: 046483 -7482WO 1(04099)

[0210] The intracellular signaling domains described herein can be combined with any of the costimulatory signaling domains described herein, any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in the CAR. For example, in some embodiments, the CAR comprises a CD28 costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO: 56, a 4-1BB costimulatory signaling domain comprising the amino acid sequence SEQ ID NO: 55 and a CD3 zeta intracellular signaling domain comprising the amino acid sequence SEQ ID NO: 59 or 60. In some embodiments, the CAR comprises (from N-ter to C-ter) an MSLN or CEACAM5 binding domain ( / or other antigen binding domain), which is linked to a cassette comprising a CD28 transmembrane domain, a CD28 costimulatory signaling, a 4-1BB costimulatory signaling domain, and a CD3 zeta intracellular signaling domain. In one embodiment, the cassette encodes a fusion protein comprises the amino acid sequence of SEQ ID NO: 75 or comprises the nucleotide sequence of SEQ ID NO: 76.

[0211] Tolerable variations of the intracellular domain will be known to those of skill in the art, while maintaining specific activity. For example, in some embodiments the intracellular domain comprises an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the costimulatory or intracellular signaling domains described in Table 1.

[0212] In some embodiments, the MSLN or CEACAM5 CAR may further comprise a spacer domain between the extracellular domain and the transmembrane domain of the CAR, and / or between the transmembrane domain and an intracellular domain of the CAR. As used herein, the term “spacer domain” generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the intracellular domain in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, e.g., 10 to 100 amino acids, or 25 to 50 amino acids. In some embodiments, the spacer domain may be a short oligo- or polypeptide linker, e.g., between 2 and 10 amino acids in length. For example, a glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain of the CAR.

[0213] Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers such as (GS)n, GSG, (GSGGS)n (SEQ ID NO: 137), (GGGS)n (SEQ ID NO: 138), and (GGGGS)n (SEQ ID NO: 139), where n represents an integer of at least 1. Attorney Docket No: 046483 -7482WO 1(04099)

[0214] Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 140), GGSGG (SEQ ID NO: 141), GSGSG (SEQ ID NO: 142), GSGGG (SEQ ID NO: 143), GGGSG (SEQ ID NO: 144), GSSSG (SEQ ID NO: 145), GGGGS (SEQ ID NO: 146), (G4S)3(SEQ ID NO: 147), (G4S)4(SEQ ID NO: 148), and the like. Those of skill in the art would be able to select the appropriate spacer sequence for use in the present invention.

[0215] The MSLN or CEACAM5 binding domains described herein may be combined with any of the transmembrane domains, any of the costimulatory signaling domains, any of the intracellular signaling domains, or any of the other domains described herein that may be included in a CAR of the present invention.

[0216] In an embodiment, an anti-MSLN CAR comprises an MSLN antigen binding domain, a hinge region, a transmembrane domain, a first costimulatory signaling domain, and an intracellular signaling domain, wherein the MSLN binding domain comprises a VHH region comprising the amino acid sequence of SEQ ID NO: 28 or 30; an IgG4 hinge region comprising the amino acid sequence of SEQ ID NO: 48; a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 43; a CD28 costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO: 56; a 4-1BB costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO: 55; and a CD3 zeta intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 59 or 60, wherein the CAR specifically binds to MSLN. In another embodiment, the anti-MSLN CAR comprises the amino acid sequence set forth in SEQ ID NO: 82 or 84 and / or is encoded by the nucleotide sequence of SEQ ID NO: 94 or 96, respectively.

[0217] In another embodiment, the anti-CEACAM5 CAR comprises a CEACAM5 antigen binding domain, a hinge region, a transmembrane domain, a first costimulatory signaling domain, a second costimulatory signaling domain, and an intracellular domain, wherein the CEACAM5 binding domain comprises a VHH region comprising the amino acid sequence of SEQ ID NO: 36; an IgG4 hinge region comprising the amino acid sequence of SEQ ID NO: 48; a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 52; a CD28 costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO: 56; a 4-1BB costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO: 55; and a CD3 zeta intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 59 or 60, wherein the CAR specifically binds to CEACAM5. In another embodiment, the Attorney Docket No: 046483 -7482WO 1(04099)

[0218] anti-CEACAM5 CAR comprises the amino acid sequence of SEQ ID NO: 91 and / or is encoded by the nucleotide sequence of SEQ ID NO: 103 or 105.

[0219] Bispecific CARs

[0220] In a further aspect, the present invention provides a bispecific CAR. In one embodiment, the bispecific CAR binds mesothelin and another tumor-associated antigen (TAA), wherein the CAR comprises an MSLN binding domain and a TAA binding domain. In another embodiment, the bi specific CAR binds CEACAM5 and another tumor-associated antigen (TAA), wherein the CAR comprises a CEACAM5 binding domain and a TAA binding domain.

[0221] In some embodiments, the bispecific CAR is a Split Bi-CAR (sbiCAR). In other embodiments, the bispecific CAR is a Tandem Bi-CAR (tbiCAR).

[0222] In certain embodiments, the tumor associated antigen is overexpressed in a solid tumor. Exemplary tumor associated antigens include, but are not limited to, alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA, CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA / CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE- A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, or any combination thereof.

[0223] In certain embodiments, the additional antigen binding domain targets a tumor associated antigen, which is a B cell antigen expressed in a non-solid tumor. Exemplary B cell antigens include but are not limited to CD5, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD33 (IL3Ra), CD34, CD37, CD38, CD40, CD52, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269. Flt3, ROR1, BCMA, FcRn5, FcRn2, CS-L CXCR4, 5, 7, IL-7 / 3R, IL7 / 4 / 3R, and L4R, and variants thereof.

[0224] In an embodiment, the bispecific CAR comprises an anti-MSLN binding domain and an anti-CEACAM5 binding domain. Attorney Docket No: 046483 -7482WO 1(04099)

[0225] In an embodiment, the Split Bi-CAR (sbiCAR) is expressed on a cell as two polypeptide chains as depicted in FIG. 16A. The two polypeptide chains may be expressed from a single transcriptional unit or from two independent transcriptional units. In one embodiment the sbiCAR is expressed from a single transcriptional unit co-expressing MSLN and CEACAM5 binding domains from an mRNA expressing a polypeptide containing an E2a self-cleaving peptide as further described herein. In certain embodiments, the sbiCAR comprises an amino acid sequence set forth in any one of SEQ ID NOs: 108-111 and / or is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 116-119, respectively. In one embodiment, the sbiCAR comprises an amino acid sequence set forth in SEQ ID NO: 110 or 111, or is encoded by the nucleotide sequence 118 or 119, respectively.

[0226] In an embodiment, the Tandem Bi-CAR (tbiCAR) is expressed on a cell as a single polypeptide chain as depicted in FIG. 16B. In this case, the two antigen binding domains are arranged in a head to tail manner from a single mRNA. In certain embodiments, the tbiCAR comprises an amino acid sequence set forth in any one of SEQ ID NOs: 112-115 and / or is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 120-123, respectively. In one embodiment, the tbiCAR comprises an amino acid sequence set forth in SEQ ID NO: 114 or 115, or is encoded by the nucleotide sequence 122 or 123, respectively.

[0227] Modified Immune Cells

[0228] The present invention provides a modified immune cell or precursor cell thereof (e.g., a modified T cell, a modified NK cell, a gamma-delta T cell) comprising an MSLN CAR, CEACAM5 CAR or bispecfic MSLN / CEACAM5 CAR described herein. Accordingly, such modified cells possess the specificity directed by the CAR expressed therefrom. For example, a modified cell of the present invention comprising an MSLN CAR possesses specificity for a target cell expressing MSLN, CEACAM5, or both.

[0229] Any modified cell comprising a CAR may be envisioned to comprise any antigen binding domain, any hinge, any transmembrane domain, any intracellular costimulatory signaling domain, and any intracellular signaling domain described herein, and can readily be understood and made by a person of skill in the art in view of the disclosure herein.

[0230] In some embodiments, the modified cell is an immune cell or precursor cell thereof. In one embodiment, the modified cell is a T cell. In a particular embodiment, the T cell is a gamma- Attorney Docket No: 046483 -7482WO 1(04099)

[0231] delta T cell. In another embodiment, the modified immune cell is an NKT cell. In another embodiment, the modified immune cell is an NK cell.

[0232] In some embodiments, the modified immune cell is an autologous cell. In some embodiments, the modified immune cells is an allogeneic cell.

[0233] In some embodiments, a modified cell of the present invention is genetically edited to disrupt the expression of one or more endogenously expressed genes to reduce the risk of autoreactivity and / or increase the efficacy of the modified cells. In certain embodiments, the gene-edited immune cells (e.g., T cells, NK cells) have a reduction, deletion, elimination, knockout, or disruption in expression of one or more endogenously expressed receptor. In one embodiment, the modified cell is genetically edited to disrupt the expression of an endogenous TCR gene product (e.g., gene products of T Cell Receptor Alpha Constant (TRAC) and T Cell Receptor Beta Constant (TRBC), Beta-2-microglobulin (B2M), and Class II Major Histocompatibility Complex Transactivator (CIITA).

[0234] In certain embodiments, the modified cell of the present disclosure is genetically edited to disrupt the expression of endogenous PD-1 gene products. In certain embodiments, disrupting the expression of endogenous PD-1 may create “checkpoint” resistant modified cells, resulting in increased tumor control. Checkpoint resistant modified cells may also be created by disrupting the expression of, for example, without limitation, the Adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), the B and T Lymphocyte Attenuator protein (BTLA / CD272), CD96, the Cytotoxic T-Lymphocyte Associated protein 4 (CTLA-4 / CD152), Indoleamine 2,3-dioxygenase (IDO), the Killer-cell Immunoglobulin-like Receptor (KIR), the Lymphocyte Activation Gene-3 (LAG3), the T cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), or the V-domain Ig suppressor of T cell activation (VISTA).

[0235] Nucleic Acids

[0236] In another aspect, the present invention provides a nucleic acid encoding any of the CARs described herein, such as those comprising an MSLN and / or CEACAM5 antigen binding domain, a transmembrane domain, an intracellular domain, and optionally a hinge domain. It should be understood that where an amino acid sequence is identified by a SEQ ID NO:, the disclosure should be further construed as including its corresponding nucleic acid, such as Attorney Docket No: 046483 -7482WO 1(04099)

[0237] described in Table 1 below. For example, in an exemplary embodiment, the MSLN antigen binding domain of the present invention comprising the amino acid sequence set forth in any one of SEQ ID NOs: 28-35 corresponds to the nucleotide sequence set forth in any one of SEQ ID NOs: 37-44 in Table 1. Likewise, in another exemplary embodiment, the CEACAM5 antigen binding domain of SEQ ID NO: 36 corresponds to the nucleotide sequence of SEQ ID NO: 45 in Table 1. Generally, the nucleic acid is provided for producing or expressing the MSLN CAR, CEACAM5 CAR or bispecific MSLN / CEACAM5 CAR as described herein, e.g., in a mammalian cell.

[0238] In some embodiments, a nucleic acid of the present disclosure further comprises a leader sequence encoding a signal peptide at the N-terminal end of the CAR and / or other encoded proteins, which is cleaved from the antigen binding domain during cellular processing and localization of the CAR and / or marker protein to the cellular membrane. Suitable signal peptide sequences are known to those of skill in the art. In an exemplary embodiment, the nucleic acid of SEQ ID NO: 61 encodes a CD8a signal peptide comprising the amino acid sequence of SEQ ID NO: 47.

[0239] In some embodiments, the nucleic acid is operably linked to a transcriptional control element, e.g., a promoter, enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art. In certain embodiments, the promoter is an EF-1 alpha promoter. An exemplary EF-1 alpha comprises the nucleotide sequence of SEQ ID NO: 130. In other embodiments, the promoter is a Rous sarcoma virus (RSV) promoter. An exemplary RSV promoter comprises the nucleotide sequence of SEQ ID NO: 131. Other constitutive promoter sequences may also be used, including, but not limited to a cytomegalovirus (CMV) immediate early promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.

[0240] Other suitable promoters include, but are not limited to cell-type specific promoters, including light and / or heavy chain immunoglobulin gene promoters, a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, an NK cell-specific promoter, and various art-known tissue specific promoters. Attorney Docket No: 046483 -7482WO 1(04099)

[0241] Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to tetracycline regulated promoters, (e.g., promoter systems including Tet Activators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, glucocorticoid promoters, progesterone promoters, and thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

[0242] In some embodiments, the locus or construct or transgene containing the suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, find use in generating irreversibly switched promoters and are well known in the art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0243] In some embodiments, the nucleic acid comprises a polynucleotide comprising a nucleotide sequence encoding a chimeric receptor inducible expression cassette. In one embodiment, the chimeric receptor inducible expression cassette is for the production of a transgenic polypeptide product that is released upon chimeric receptor signaling. See, e.g., Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Attorney Docket No: 046483 -7482WO 1(04099)

[0244] Immunotherapy (2015) 7(5): 535-544. In some embodiments, a nucleic acid of the present disclosure comprises one or more transgenes operably linked to a T-cell activation responsive promoter (e.g., NF AT).

[0245] In some embodiments, the nucleic acid may additionally encode one or more additional protein(s) of interest, including but not limited to, a cytokine, a cytokine antagonist, an immune checkpoint inhibitor, a chemokine, a switch receptor, a marker protein, or a combination thereof.

[0246] Exemplary cytokines include, but are not limited to, IL-2, IL-7, IL-9, IL- 12, IL- 15, IL- 18, IL-21, IL-22, and IL-23.

[0247] Exemplary cytokine antagonists include, but are not limited to, those inhibiting cytokine release syndrome (CRS), such as anti-IL-6, anti-IL-1, anti-IFN-y, anti-TNFa, anti-IL-8, anti-IL10, anti-GM-CSF, including scFvs and / or nanobodies thereof.

[0248] Exemplary immune checkpoint inhibitor include, but are not limited to, antagonists of a protein selected from the group consisting of the Programmed Death 1 receptor (PD-1), the Adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), the B and T Lymphocyte Attenuator protein (BTLA / CD272), CD96, the Cytotoxic T-Lymphocyte Associated protein 4 (CTLA-4 / CD152), Indoleamine 2,3 -dioxygenase (IDO), the Killer-cell Immunoglobulin-like Receptor (KIR), the Lymphocyte Activation Gene-3 (LAG3), the T cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), the V-domain Ig suppressor of T cell activation, and combinations thereof.

[0249] Exemplary immune checkpoint inhibitors include, but are not limited to PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFRbeta.

[0250] Exemplary chemokines include, but are not limited to CXCL9 and CCL19.

[0251] The term “switch receptor” refers to a molecule designed to switch a negative signal transduction signal into a positive signal. In some embodiments, the switch receptor is a chimeric protein comprising a first protein or fragment thereof associated with a negative signal, and a second protein or fragment thereof associated with a positive signal. Examples of proteins associated with a negative signal include, without limitation, CTLA-4, PD-1, BTLA, TIM-3 and the like. Examples of proteins associated with a positive signal include, without limitation, CD28, ICOS, 4-1BB, IL-12R, an extracellular domain of a TGFβR (e.g, TGFβRI or TGFβRII) and the like. Exemplary switch receptors include, but are not limited to, PD1-CD28, TIM3-CD28, PD1-4-1BB, PD1A132L-4-1BB, PD1A132L-CD28, TGFβRI-IL-12Rβ1, TGFβRI-IL-12Rβ2, Attorney Docket No: 046483 -7482WO 1(04099)

[0252] and the dominant-negative TGFpRII switch receptor, TGFβRIIDN Other suitable switch receptors for use in the present invention are described in US Patent No. 10,981,969 B2, the disclosure of which is incorporated herein by reference.

[0253] Exemplary markers (or reporters) include, but are not limited to, minimal low affinity nerve growth factor (mLNGFR), truncated LNGFR (tLNGFR), truncated EGFR (tEGFR), truncated CD 19 (tCD19), truncated CD34 (tCD34), blue fluorescent protein (BFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and an combination thereof.

[0254] In some embodiments, the nucleic acid comprises one or more polynucleotides comprising nucleotide sequence(s) encoding a bispecific CAR, or co-expressing one or more additional CARs with the MSLN CAR, CEACAM5 CAR, or bispecific MSLN / CEACAM5 CAR of the present invention. In certain embodiments, the bispecific CAR or additional CAR includes an antigen binding domain targeting a tumor associated antigen. In certain embodiments, the tumor associated antigen is overexpressed in a solid tumor. Exemplary tumor associated antigens include, but are not limited to, alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA, CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA / CEACAM5, claudin 18.2, c-Met, DLL3, MSLN, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypi can-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

[0255] In certain embodiments, the additional CAR(s) include an antigen binding domain targeting a tumor associated antigen B cell antigen expressed in a non-solid tumor. Exemplary B cell antigens include but are not limited to CD5, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD33 (IL3Ra), CD34, CD37, CD38, CD40, CD52, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269. Flt3, ROR1, BCMA, FcRn5, FcRn2, CS-L CXCR4, 5, 7, IL-7 / 3R, IL7 / 4 / 3R, and L4R, and variants thereof. Attorney Docket No: 046483 -7482WO 1(04099)

[0256] In embodiments comprising or expressing an additional CAR, any of the CAR components described herein may be used in a similar manner for the additional CAR(s).

[0257] In some embodiments, the additional protein(s) of interest is / are operatively linked to a different promoter than the one directing expression of the MSLN CAR, CEACAM5 CAR, or MSLN / CEAM5 bi-CAR. In other embodiments, the additional protein(s) is / are co-expressed with the MSLN CAR, CEACAM5 CAR, or MSLN / CEAM5 bi-CAR from a common promoter using self-cleaving 2A peptides fused in-frame between two or more polypeptides using an internal ribosome entry site (IRES) between two or more polypeptides.

[0258] As used herein, a “self-cleaving 2A peptide” or “2A peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine tescho virus- 1 (PTV-1); and cardioviruses such as Theilovirus and encephalomyocarditis viruses. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.

[0259] In one embodiment, the nucleic acid of the present disclosure encodes the T2A selfcleaving peptide of SEQ ID NO: 160. In another embodiment, the nucleic acid encodes the P2A self-cleaving peptide of SEQ ID NO: 161. In another embodiment, the nucleic acid encodes the E2A self-cleaving peptide of SEQ ID NO: 162. In another embodiment, the nucleic acid encodes the F2A self-cleaving peptide of SEQ ID NO: 163. Those of skill in the art would be able to select the appropriate self-cleaving peptide for use in the present invention.

[0260] In some embodiments, a polynucleotide comprising an internal ribosome entry site (IRES) is interposed between two or more polypeptides. As used herein, “an internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a protein coding region, thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those of skill in the art, including, without limitation, IRES obtainable from viral or cellular mRNA sources, e.g., immunoglobulin heavy-chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translational initiation factor eIF4G; yeast Attorney Docket No: 046483 -7482WO 1(04099)

[0261] transcription factors TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). An exemplary IRES, encephalomyocarditis virus (EMCV) IRES, comprises the nucleotide sequence of SEQ ID NO: 164. Those of skill in the art would be able to select the appropriate IRES for use in the present invention.

[0262] A nucleic acid of the present disclosure may be present within an expression vector and / or a cloning vector.

[0263] Expression Vectors and Expression Constructs

[0264] In another aspect, the present invention provides a cloning vector, expression vector or expression construct comprising a nucleic acid encoding a targeting protein (e.g., anti-MSLN targeting protein) described herein. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example, and should not be construed in any way as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif, USA); pTrc99A, pKK223-3, pKK233-3, pDR40, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0265] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Set. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Set. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski Attorney Docket No: 046483 -7482WO 1(04099)

[0266] et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); a retroviral vector e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0267] Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.

[0268] In certain embodiments, the vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U. S. Pat. No. 6,326,193).

[0269] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used for expressing the MSLN CAR, CEACAM5 CAR, MSLN / CEACAM5 bi-CAR, and / or any other additional transgenes. In some embodiments, the expression vector (e.g., lentiviral vector) will comprise additional elements that will aid in the functional expression of the targeting protein or targeting protein encoded therein. In some embodiments, an expression vector comprises a mammalian promoter. In one embodiment, the vector comprises an elongation-factor- 1 -alpha (EF-la) promoter. Use of an EF-la promoter may increase the efficiency in expression of downstream transgenes, such as the nucleic acid encoding the targeting protein. Physiologic promoters (e.g., an EF-la promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of the retroviral vector to transform stem cells Other physiological promoters suitable for use in a vector (e.g., a lentiviral vector) are known to those of skill in the art and may be incorporated into a vector of the present invention. Attorney Docket No: 046483 -7482WO 1(04099)

[0270] In some embodiments, the vector (e.g., a lentiviral vector) further comprises a nonrequisite c / .s-acting sequence that may improve titers and gene expression. One non-limiting example of a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS) which is important for efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention.

[0271] In some embodiments, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression, and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Accordingly, in some embodiments a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g., a lentiviral vector) of the present invention.

[0272] A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and to viral replication. In one embodiment, a vector (e.g., lentiviral vector) of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector e.g., lentiviral vector) of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector (e.g., lentiviral vector) of the present invention may comprise a WPRE sequence, cPPT sequence, RRE sequence, 5 ’LTR, and 3’ U3 deleted LTR’ in addition to a nucleic acid encoding for targeting protein (e.g., anti-MSLN targeting protein).

[0273] Vectors of the present invention may be self-inactivating vectors. As used herein, the term “self-inactivating vector” refers to vectors in which the 3’ LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector may prevent viral transcription beyond the first round of viral replication. Consequently, a selfinactivating vector may be capable of infecting and then integrating into a host genome (e.g., a Attorney Docket No: 046483 -7482WO 1(04099)

[0274] mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors may greatly reduce the risk of creating a replication-competent virus.

[0275] In one embodiment, the lentiviral vector is a self-inactivating HIV vector comprising a 5’ U3 LTR (e.g., SEQ ID NO: 132), a 3’ U3 deleted LTR (e.g., SEQ ID NO: 136), a partial HIV gag sequence comprising an RRE sequence and packaging signal (r|r ) (e.g., SEQ ID NO: 133), and / or an HIV central polypurine tract (cPPT) sequence (SEQ ID NO: 134). In some embodiments, the expression vector and / or HIV vector comprises a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE). In an exemplary embodiment, the WPRE comprises the nucleotide sequence of SEQ ID NO: 135.

[0276] In some embodiments, a nucleic acid encoding the MSLN CAR, CEAC AM5 CAR or MSLN / CEACAM5 bi-CAR comprises a lentivirus vector. In one embodiment, a nucleic acid encoding a CEACAM5 CAR comprises a lentivirus expression construct comprising a nucleotide sequence as set forth in e.g., SEQ ID NO: 124 or 125. In other embodiments, the nucleic acid encoding an MSLN / CEACAM5 bi-CAR comprises a lentivirus expression construct comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 126-129.

[0277] In order to assess the expression of a polypeptide or portions thereof, the expression vector or construct to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes.

[0278] Reporter genes may be used e.g., for tracking T and NK cells expressing the MSLN CARs as described herein. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., flow cytometry or enzymatic activity.

[0279] Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells.

[0280] In some embodiments, a nucleic acid of the present invention may be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; Attorney Docket No: 046483 -7482WO 1(04099)

[0281] any known method can be used to synthesize RNA comprising a sequence encoding a chimeric receptor of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a chimeric receptor of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a chimeric receptor of the present disclosure.

[0282] Nucleotide and amino sequences of exemplary MSLN binding domains, CEACAM5 biding domain, CAR components, expression constructs, sequence elements, and primers can be found in Table 1.

[0283] Table 1: Nucleotide and Amino Acid Sequences.

[0284] SEQ Description Sequence

[0285] ID NO:

[0286] 1 MSLN VHH1 CDR1 GFTFSMSAMN

[0287] 2 MSLN VHH1 CDR2 GITNSGGFTS

[0288] 3 MSLN VHH1 CDR3 TRFASSLSPPVS

[0289] 4 MSLN VHH10 CDR1 ERTFSTYALGW

[0290] 5 MSLN VHH10 CDR2 AIGYSHAETH

[0291] 6 MSLN VHH10 CDR3 AARPSPMATLSAQLYDY

[0292] 7 MSLN VHH12 CDR1 RSIFSTYAMG

[0293] 8 MSLN VHH12 CDR2 AIGYSHGNTD

[0294] 9 MSLN VHH12 CDR3 AAYTGPRPPRQAPLYDY

[0295] 10 MSLN VHH16 CDR1 GFTFSMSTMN

[0296] 11 MSLN VHH16 CDR2 GINNSGGFTS

[0297] 12 MSLN VHH16 CDR3 TRFASSLSPPVS

[0298] 13 MSLNVHH18 CDR1 ERTFSTYAMA

[0299] 14 MSLNVHH18 CDR2 ATEYSVAVTT

[0300] 15 MSLN VHH18 CDR3 AARPSPMATSSAQTYEY

[0301] 16 MSLNVHH61 CDR1 ERIFSTYAQG

[0302] 17 MSLN VHH61 CDR2 SIGYSATDTR

[0303] 18 MSLNVHH61 CDR3 AGRPSPVPTSSAQLYDY

[0304] 19 MSLN VHH67 CDR1 ERTFSTYAMG

[0305] 20 MSLN VHH67 CDR2 ATGYSHAMTN

[0306] 21 MSLN VHH67 CDR3 AARPSPMATLSAQTYDY

[0307] 22 MSLN VHH68 CDR1 GRIFGNYAMG

[0308] 23 MSLN VHH68 CDR2 AIAYNFDTK

[0309] 24 MSLN VHH68 CDR3 AQRSGPGFSDNLRLYDY

[0310] 25 VHHB30 HCDR1 INAMG

[0311] 26 VHHB30 HCDR2 TITNGVNGVTTNYANSVKGR

[0312]

[0313] 27 VHHB30 HCDR3 TPDPLRYLFDS Attorney Docket No: 046483 -7482WO 1(04099)

[0314] Anti-MSLN VHH1 AA QVQLQESGGGSVQPGGSLRLSCAASGFTFSMSAMNWVRQTAG KGLEWVSGITNSGGFTSYADSVKGRFTISRDNAKNTLYLQMNS LKPEDTAVYYCTRFASSLSPPVSQGTQVTVSS

[0315] Anti-MSLN VHH10 QVQLQESGGGLVQAGGSLRLSCAASERTFSTYALGWFRQAPGK AA EREFVAAIGYSHAETHYADSVKGRFTISRDNAKNTVYLQMNSL QSYDTALYFCAARPSPMATLSAQLYDYWGQGTQVTVSS

[0316] Anti-MSLN VHH12 QVQLQESGGGLVQTGGSLRLSCAASRSIFSTYAMGWFRQAPGK AA EREFVAAIGYSHGNTDYVDSVKGRFTISRDNAKNTMYLQMNSL RPEDTAVYYCAAYTGPRPPRQAPLYDYWGQGTQVTVSS

[0317] Anti-MSLN VHH16 QVQLQESGGGSVQPGGSLRLSCAASGFTFSMSTMNWVRQTPG AA KGLEWVSGINNSGGFTSYADSVKGRFTISRDNAKNMLYLQMNS LKPEDTAVYYCTRFASSLSPPVSQGTQVTVSS

[0318] Anti-MSLN VHH18 QVQLQESGGGLVQAGGSLRLSCAASERTFSTYAMAWFRQAPG AA KEREFVAAIEYSVAVTTYADSVKGRFTISRDIAQNTVYLQMNSL KPDDTAVYFCAARPSPMATSSAQTYEYWGQGTQVTVSS

[0319] Anti-MSLN VHH61 QVQLQESGGGLVQVGDSLRLSCAASERIFSTYAQGWFRQAPGK AA EREFVASIGYSATDTRYADSVKGRFTISRDSAKNTIDLLMNSLK PDDTAVYYCAGRPSPVPTSSAQLYDYWGQGTQVTVSS

[0320] Anti-MSLN VHH67 QVQLQESGGGLVQAGGSLRLSCAASERTFSTYAMGWFRQAPG AA KEREFVAAIGYSHAMTNYADSVKGRFTISRDNAKNTVYLQMN SLKSYDTAVYFCAARPSPMATLSAQTYDYWGQGTQVTVSS

[0321] Anti-MSLN VHH68 QVQLQESGGGVVQAGGSLRLSCVGSGRIFGNYAMGWFRQAPG AA KEREFVAAIAYNFDTKYADSVKGRFTISRDNARNTLYLQMNNL KPEDTAVYYCAQRSGPGFSDNLRLYDYWGQGTQVTVSS

[0322] Anti-CEACAM5 QVQLQESGGGLVQAGGSLKLSCAASGSIHSINAMGWYRQTPGN VHHB30 AA QRELVATITNGVNGVTTNYANSVKGRFTISRDNAKNTVYLQM NSLKPEDTAVYYCTPDPLRYLFDSWGQGTQVTVSS

[0323] Anti-MSLN VHH1 CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGCC DNA TGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCAC CTTCAGTATGTCTGCCATGAACTGGGTCCGCCAAACTGCAGG CAAGGGGCTCGAGTGGGTCTCCGGTATTACTAATAGTGGTGG ATTTACAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCAT CTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGA ATAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTACA AGATTTGCTAGTTCGTTATCTCCACCCGTGAGCCAGGGGACC CAGGTCACCGTCTCCTCA

[0324] Anti-MSLN VHH10 CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGC DNA TGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGAACCCAC CTTCAGTACGTATGCCTTGGGCTGGTTCCGCCAGGCTCCAGG AAAGGAGCGTGAATTTGTAGCGGCTATTGGCTACAGTCATGC TGAGACTCACTATGCAGACTCCGTGAAGGGCCGATTCACCAT TTCCCGAGACAACGCCAAGAACACGGTGTATCTTCAAATGA ACAGCCTACAGTCATACGACACGGCCCTTTATTTTTGTGCAG CACGTCCTTCGCCAATGGCGACTTTATCCGCTCAACTCTATG ACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0325] Anti-MSLN VHH12 CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGAC DNA TGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTAGAAGTAT TTTCAGTACATATGCCATGGGCTGGTTCCGCCAGGCTCCAGG GAAGGAGCGTGAGTTTGTAGCGGCTATTGGTTATAGTCATGG

[0326]

[0327] AAACACAGACTATGTAGACTCCGTGAAGGGCCGATTCACCA Attorney Docket No: 046483 -7482WO 1(04099)

[0328] TCTCCAGAGACAACGCCAAGAACACGATGTATCTGCAAATG AACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCA GCATACACAGGACCTCGGCCACCACGGCAGGCGCCCTTATA TGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0329] Anti-MSLN VHH16 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGCC DNA TGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCAC CTTCAGTATGTCTACCATGAACTGGGTCCGCCAAACCCCAGG AAAGGGGCTCGAGTGGGTCTCCGGTATTAACAATAGTGGTG GATTTACAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCA TCTCCAGAGACAACGCCAAGAATATGCTGTATCTGCAAATG AATAGCCTGAAACCTGAGGACACGGCTGTGTATTACTGTACA AGATTTGCTAGTTCGTTATCTCCACCCGTGAGCCAGGGGACC CAGGTCACCGTCTCCTCA

[0330] Anti-MSLN VHH18 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGC DNA TGGGGGCTCTCTACGACTCTCCTGTGCAGCCTCTGAACGCAC CTTCAGTACTTATGCCATGGCCTGGTTCCGCCAGGCTCCAGG GAAGGAGCGTGAATTTGTAGCAGCTATTGAGTATAGTGTTGC AGTGACAACGTATGCAGACTCCGTCAAGGGCCGATTCACCA TCTCCAGAGACATCGCCCAGAACACGGTGTATCTGCAAATG AACAGCCTGAAACCTGACGACACGGCCGTTTATTTTTGTGCA GCACGTCCTTCGCCTATGGCGACTTCATCCGCTCAAACCTAT GAGTATTGGGGCCAGGGAACCCAGGTCACCGTCTCCTCA

[0331] Anti-MSLN VHH61 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGT DNA TGGGGACTCTCTTAGACTCTCCTGTGCAGCCTCTGAACGCAT CTTCAGTACGTATGCCCAGGGCTGGTTCCGCCAGGCTCCAGG GAAGGAGCGTGAATTTGTAGCAAGCATTGGCTATAGTGCGA CTGACACAAGATATGCAGACTCCGTGAAGGGCCGATTCACC ATCTCCAGAGACAGCGCCAAGAACACGATCGATCTACTTAT GAACAGCCTGAAACCTGACGACACGGCCGTTTATTACTGTGC AGGACGTCCTTCGCCAGTACCGACTTCATCCGCTCAACTCTA TGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0332] Anti-MSLN VHH67 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGC DNA TGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGAACGCAC CTTCAGTACGTATGCCATGGGCTGGTTCCGCCAGGCTCCAGG GAAGGAGCGTGAATTTGTAGCGGCTATTGGCTACAGTCATGC TATGACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCAT TTCCCGAGACAACGCCAAGAACACGGTGTATCTGCAAATGA ACAGCCTGAAGTCATACGACACGGCCGTTTATTTTTGTGCAG CACGTCCTTCGCCTATGGCGACTTTATCCGCTCAAACCTATG ACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0333] Anti-MSLN VHH68 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGAGTGGTGCAGGC DNA TGGGGGCTCTCTGAGACTCTCCTGTGTAGGCTCTGGACGCAT CTTCGGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCTGG AAAGGAGCGTGAGTTTGTTGCAGCAATTGCGTATAACTTCGA CACAAAGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTC CAGAGACAACGCCAGGAACACGCTGTATCTGCAAATGAACA ACCTGAAACCTGAAGACACGGCGGTTTATTACTGTGCACAA AGATCAGGGCCCGGATTTAGTGATAACCTTCGTCTGTATGAC

[0334]

[0335] TACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA Attorney Docket No: 046483 -7482WO 1(04099)

[0336] Anti-CEACAM5 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGC VHHB30 DNA TGGGGGGTCTCTAAAACTCTCCTGTGCAGCCTCTGGAAGCAT (without signal peptide) CCACAGTATCAATGCCATGGGCTGGTACCGCCAGACTCCAG GGAACCAGCGCGAGTTGGTCGCAACTATTACTAATGGTGTTA ATGGTGTCACCACAAACTATGCAAACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTA CTGTACACCAGACCCCCTGCGGTACCTGTTTGATTCCTGGGG CCAGGGGACCCAGGTCACCGTCTCCTCA

[0337] Anti-CEACAM5 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTG VHHB30 DNA (with CTGCTCCACGCCGCCAGGCCGGGATCCGCGGCCCAGGCGGC signal peptide) CCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGG CTGGGGGGTCTCTAAAACTCTCCTGTGCAGCCTCTGGAAGCA TCCACAGTATCAATGCCATGGGCTGGTACCGCCAGACTCCAG GGAACCAGCGCGAGTTGGTCGCAACTATTACTAATGGTGTTA ATGGTGTCACCACAAACTATGCAAACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTA CTGTACACCAGACCCCCTGCGGTACCTGTTTGATTCCTGGGG CCAGGGGACCCAGGTCACCGTCTCCTCA

[0338] Hu CD8 signal peptide MALPVTALLLPLALLLHAARP

[0339] Hu IgG4 hinge ESKYGPPCPPCP

[0340] Hu CD 8 hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD Hu CD28 hinge IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP Hu CD8 TM IYIWAPLAGTCGVLLLSLVITLYC

[0341] Hu CD28 TM FWVLVVVGGVLACYSLLVTVAFIIFWV

[0342] Hu ICOS TM Peptide FWLPIGCAAFVVVCILGCILI

[0343] Hu 0X40 TM Peptide VAAILGLGLVLGLLGPLAILL

[0344] Hu 4- IBB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL Costimulatory

[0345] Signaling Domain

[0346] Peptide

[0347] Hu CD28 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS Costimulatory

[0348] Signaling Domain

[0349] Peptide

[0350] Hu ICOS CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL Costimulatory

[0351] Signaling Domain

[0352] Peptide

[0353] Hu 0X40 ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI Costimulatory

[0354] Signaling Domain

[0355] Peptide

[0356] CD3 zeta Intracellular RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD Signaling domain PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK Peptide (QI 4) GHDGLYQGLSTATKDTYDALHMQALPPR

[0357] Hu CD3 zeta RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRD Intracellular Signaling PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK

[0358]

[0359] GHDGLYQGLSTATKDTYDALHMQALPPR Attorney Docket No: 046483 -7482WO 1(04099)

[0360] Domain Peptide

[0361] (Q14K)

[0362] Hu CD 8 signal peptide ATGGCTCTGCCAGTGACCGCACTGCTCCTGCCCCTGGCACTG DNA CTGCTGCACGCAGCAAGGCCA

[0363] Hu IgG4 hinge DNA GAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCT

[0364] Hu CD8 hinge DNA ACTACAACACCTGCGCCCCGACCACCAACACCCGCCCCGAC CATTGCCTCCCAACCCCTGTCCCTGAGACCCGAGGCATGCAG GCCAGCTGCAGGTGGAGCAGTGCACACCCGGGGCCTCGACT TCGCCTGTGAT

[0365] Hu CD28 hinge DNA ATCGAAGTCATGTACCCCCCTCCATATCTTGACAATGAGAAG AGCAACGGCACCATCATTCATGTGAAAGGCAAACACTTGTG TCCAAGTCCTCTGTTTCCTGGACCCTCTAAGCCC

[0366] Hu CD8 TM DNA ATCTACATTTGGGCTCCCTTGGCAGGCACCTGTGGAGTTCTG CTGCTGTCCCTCGTCATCACGCTCTATTGT

[0367] Hu CD28 TM DNA TTCTGGGTCTTGGTGGTCGTAGGAGGAGTTTTGGCGTGCTAC TCCTTGCTCGTGACCGTGGCCTTTATCATATTCTGGGTC

[0368] Hu ICOS TM DNA TTCTGGCTGCCCATTGGATGTGCTGCCTTTGTGGTCGTGTGCA TCCTTGGTTGCATCCTCATC

[0369] Hu 0X40 TM DNA GTGGCTGCTATCCTGGGCCTGGGACTCGTTCTCGGACTTCTG GGACCCCTGGCCATCTTGCTC

[0370] Hu 4- IBB AAACGCGGCAGAAAAAAGCTCCTCTATATTTTCAAGCAACC Costimulatory CTTCATGAGGCCCGTGCAGACTACACAGGAGGAAGATGGGT Signaling Domain GCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGGGGGTGTGAA DNA CTG

[0371] Hu CD28 AGAAGTAAGAGATCAAGACTCCTGCATAGTGACTACATGAA Costimulatory CATGACACCACGCAGACCCGGTCCAACGAGAAAGCACTACC Signaling Domain AGCCCTACGCACCTCCGAGAGACTTTGCCGCTTACCGGTCC DNA

[0372] Hu ICOS TGTTGGTTGACGAAGAAAAAGTACTCCTCCTCAGTGCACGAT Costimulatory CCAAATGGCGAGTACATGTTCATGCGGGCGGTCAATACCGC Signaling Domain AAAGAAAAGCCGCCTGACGGATGTTACACTT

[0373] DNA

[0374] Hu 0X40 GCCCTTTATTTGCTTCGACGCGACCAAAGACTGCCACCCGAT Costimulatory GCCCATAAACCACCCGGAGGAGGTTCCTTCAGGACCCCTATC Signaling Domain CAGGAGGAGCAGGCCGATGCTCATAGCACTCTGGCCAAGAT DNA C CD3 zeta Intracellular CGAGTGAAATTCAGTAGATCCGCCGATGCCCCCGCATACCA Signaling Domain ACAGGGACAGAACCAGCTCTACAACGAACTGAATTTGGGGA (Q14) DNA GACGGGAGGAGTACGACGTTCTTGATAAGAGAAGGGGTAGA GATCCTGAGATGGGTGGTAAACCAAGGCGCAAGAATCCCCA GGAAGGTCTCTACAACGAACTGCAGAAGGACAAGATGGCCG AAGCTTACTCCGAGATCGGAATGAAGGGAGAGCGCAGAAGG GGAAAGGGCCACGATGGGCTGTATCAAGGCCTTAGCACAGC TACAAAAGACACCTATGACGCGTTGCACATGCAGGCTCTTCC ACCCAGA

[0375] Hu CD3 zeta AGAGTGAAGTTTAGTAGAAGCGCAGATGCACCCGCATATAA Intracellular Signaling ACAGGGCCAGAACCAACTGTACAATGAATTGAATCTGGGAC Domain(Q14K) DNA GACGCGAGGAGTACGACGTGCTGGATAAACGCAGAGGACGA

[0376] GACCCAGAGATGGGCGGGAAACCACGGAGGAAGAATCCGC

[0377]

[0378] AGGAGGGCCTCTACAACGAGCTCCAGAAGGACAAGATGGCC Attorney Docket No: 046483 -7482WO 1(04099)

[0379] GAGGCCTATTCCGAGATTGGCATGAAGGGGGAGAGGAGAAG AGGTAAGGGGCACGACGGACTTTACCAGGGACTCAGTACCG CGACCAAGGACACCTATGATGCCCTGCATATGCAAGCGCTCC CTCCCCGG

[0380] 28BBz Protein FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNM CD28TM-CD28 TPRRPGPTRKHYQPYAPPRDFAAYRSASKRGRKKLLYIFKQPFM costimulatory 4- IBB RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQ costimulatory CD3 NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY gamma signaling NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR

[0381] 28BBz DNA TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTAT CD28TM-CD28 AGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGG costimulatory 4- IBB AGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACAT costimulatory CD3 zeta GACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCC signaling CTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGCTAG CAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAAC CATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGC TGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGA ACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGT ACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTA GGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGG CCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAAC CCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCC GGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT ACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC CCTGCCCCCTCGCTAA MSLN precursor MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQE protein AAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVAL AQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSG PQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLL SEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQ EAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIP QGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKA REIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVL KHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLK ALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLD TLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVL YPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMD LATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRD WILRQRQDDLDTLGLGLQGG1PNGYLVLDLSMQEALSGTPCLL GPGPVLTVLALLLASTLA MSLN mature protein MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTEVEKTACP SGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTY EQLDVLKHKLDELYPQGYPESV1QHLGYLFLKMSPEDIRKWNV TSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQL DKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDP RQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQ

[0382]

[0383] QNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEER Attorney Docket No: 046483 -7482WO 1(04099)

[0384] HRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALS GTPCLLGPGPVLTVLALLLASTLA MSLN Del-RI peptide MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTSLETLKAL LEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTL TAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYP KARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLA TFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWIL RQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPG PVLTVLALLLASTLA MSLN Del-RII peptide MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTEVEKTACP SGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTY EQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNV TYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLP LTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGL GLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLAST LA MSLN Del-RIII MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTEVEKTACP peptide SGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTY EQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNV TSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQL DKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDP RQLDVLYPKARLAFQNMNGSEGTPCLLGPGPVLTVLALLLAST LA MSLN VHH1.28BBz QVQLQESGGGSVQPGGSLRLSCAASGFTFSMSAMNWVRQTAG CAR protein KGLEWVSGITNSGGFTSYADSVKGRFTISRDNAKNTLYLQMNS LKPEDTAVYYCTRFASSLSPPVSQGTQVTVSSAAATSGQAGQES KYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRL LHSDYMNMTPRRPGPTSTRKHYQPYAPPRDFAAYRSKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR MSLN VHH10.28BBz VQLQESGGGLVQAGGSLRLSCAASERTFSTYALGWFRQAPGKE CAR protein REFVAAIGYSHAETHYADSVKGRFTISRDNAKNTVYLQMNSLQ SYDTALYFCAARPSPMATLSAQLYDYWGQGTQVTVSSAAATS GQAGQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTSTRKHYQPYAPPRDFAAYR SKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR MSLN VHH12.28BBz QVQLQESGGGLVQTGGSLRLSCAASRSIFSTYAMGWFRQAPGK CAR protein EREFVAAIGYSHGNTDYVDSVKGRFTISRDNAKNTMYLQMNSL RPEDTAVYYCAAYTGPRPPRQAPLYDYWGQGTQVTVSSAAAT SGQAGQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIF WVRSKRSRLLHSDYMNMTPRRPGPTSTRKHYQPYAPPRDFAA YRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG

[0385]

[0386] KGHDGLYQGLSTATKDTYDALHMQALPPR Attorney Docket No: 046483 -7482WO 1(04099)

[0387] MSLN VHH16.28BBz QVQLQESGGGSVQPGGSLRLSCAASGFTFSMSTMNWVRQTPG CAR protein KGLEWVSGINNSGGFTSYADSVKGRFTISRDNAKNMLYLQMNS LKPEDTAVYYCTRFASSLSPPVSQGTQVTVSSAAATSGQAGQES KYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRL LHSDYMNMTPRRPGPTSTRKHYQPYAPPRDFAAYRSKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR MSLN VHH18.28BBz QVQLQESGGGLVQAGGSLRLSCAASERTFSTYAMAWFRQAPG CAR protein KEREFVAAIEYSVAVTTYADSVKGRFT1SRDIAQNTVYLQMNSL KPDDTAVYFCAARPSPMATSSAQTYEYWGQGTQVTVSSAAAT SGQAGQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIF WVRSKRSRLLHSDYMNMTPRRPGPTSTRKHYQPYAPPRDFAA YRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR MSLN VHH61.28BBz QVQLQESGGGLVQVGDSLRLSCAASERIFSTYAQGWFRQAPGK CAR protein EREFVASIGYSATDTRYADSVKGRFTISRDSAKNTIDLLMNSLK PDDTAVYYCAGRPSPVPTSSAQLYDYWGQGTQVTVSSAAATS GQAGQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTSTRKHYQPYAPPRDFAAYR SKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR MSLN VHH67.28BBz QVQLQESGGGLVQAGGSLRLSCAASERTFSTYAMGWFRQAPG CAR protein KEREFVAAIGYSHAMTNYADSVKGRFTISRDNAKNTVYLQMN SLKSYDTAVYFCAARPSPMATLSAQTYDYWGQGTQVTVSSAA ATSGQAGQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTSTRKHYQPYAPPRDFAA YRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR MSLN VHH68.28BBz QVQLQESGGGVVQAGGSLRLSCVGSGRIFGNYAMGWFRQAPG protein CAR protein KEREFVAAIAYNFDTKYADSVKGRFTISRDNARNTLYLQMNNL KPEDTAVYYCAQRSGPGFSDNLRLYDYWGQGTQVTVSSAAAT SGQAGQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIF WVRSKRSRLLHSDYMNMTPRRPGPTSTRKHYQPYAPPRDFAA YRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR VHHB30-30BBz CAR QVQLQESGGGLVQAGGSLKLSCAASGSIHSINAMGWYRQTPGN protein (no signal QRELVATITNGVNGVTTNYANSVKGRFTISRDNAKNTVYLQM peptde) NSLKPEDTAVYYCTPDPLRYLFDSWGQGTQVTVSSAAATSSGE

[0388] SKYGPPCPPCPDIFWVLVVVGGVLACYSLLVTVAFIIFWVSKRG

[0389]

[0390] RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS Attorney Docket No: 046483 -7482WO 1(04099)

[0391] RSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR VHHB30-28BBz CAR QVQLQESGGGLVQAGGSLKLSCAASGSIHSINAMGWYRQTPGN protein (3rd gen, no QRELVATITNGVNGVTTNYANSVKGRFTISRDNAKNTVYLQM signal peptide) NSLKPEDTAVYYCTPDPLRYLFDSWGQGTQVTVSSAAATSSGE SKYGPPCPPCPDIFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSR SADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR VHHB30-30BBz CAR MALPVTALLLPLALLLHAARPGSAAQAAQVQLQESGGGLVQA protein GGSLKLSCAASGSIHSINAMGWYRQTPGNQRELVATITNGVNG VTTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTP DPLRYLFDSWGQGTQVTVSSAAATSSGESKYGPPCPPCPDIFWV LVVVGGVLACYSLLVTVAFIIFWVSKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR VHHB30-28BBz CAR MALPVTALLLPLALLLHAARPGSAAQAAQVQLQESGGGLVQA protein (3rd gen) GGSLKLSCAASGSIHSINAMGWYRQTPGNQRELVATITNGVNG VTTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTP DPLRYLFDSWGQGTQVTVSSAAATSSGESKYGPPCPPCPDIFWV LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSASKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR MSLN VHH1.28BBz CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGCC CAR DNA TGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCAC CTTCAGTATGTCTGCCATGAACTGGGTCCGCCAAACTGCAGG CAAGGGGCTCGAGTGGGTCTCCGGTATTACTAATAGTGGTGG ATTTACAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCAT CTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGA ATAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTACA AGATTTGCTAGTTCGTTATCTCCACCCGTGAGCCAGGGGACC CAGGTCACCGTCTCCTCAGCGGCCGCCACTAGTGGCCAGGCC GGCCAGGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCT TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTAT AGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGG AGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACAT GACTCCCCGCCGCCCCGGGCCCACTAGTACCCGCAAGCATTA CCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAAC CATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGC TGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGA

[0392]

[0393] ACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGT Attorney Docket No: 046483 -7482WO 1(04099)

[0394] ACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTA GGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGG CCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAAC CCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCC GGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT ACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC CCTGCCCCCTCGCTAA MSLN VHH10.28BBz CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGC CAR DNA TGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGAACCCAC CTTCAGTACGTATGCCTTGGGCTGGTTCCGCCAGGCTCCAGG AAAGGAGCGTGAATTTGTAGCGGCTATTGGCTACAGTCATGC TGAGACTCACTATGCAGACTCCGTGAAGGGCCGATTCACCAT TTCCCGAGACAACGCCAAGAACACGGTGTATCTTCAAATGA ACAGCCTACAGTCATACGACACGGCCCTTTATTTTTGTGCAG CACGTCCTTCGCCAATGGCGACTTTATCCGCTCAACTCTATG ACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCG GCCGCCACTAGTGGCCAGGCCGGCCAGGAGAGCAAGTACGG CCCTCCCTGCCCCCCTTGCCCTTTTTGGGTGCTGGTGGTGGTT GGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCC TTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTG CACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCC ACTAGTACCCGCAAGCATTACCAGCCCTATGCCCCACCACGC GACTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGAAACT CCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAAC TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAG AAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGC TCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGAT GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATG AACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATG GCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA MSLN VHH12.28BBz CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGAC CAR DNA TGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTAGAAGTAT TTTCAGTACATATGCCATGGGCTGGTTCCGCCAGGCTCCAGG GAAGGAGCGTGAGTTTGTAGCGGCTATTGGTTATAGTCATGG AAACACAGACTATGTAGACTCCGTGAAGGGCCGATTCACCA TCTCCAGAGACAACGCCAAGAACACGATGTATCTGCAAATG AACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCA GCATACACAGGACCTCGGCCACCACGGCAGGCGCCCTTATA TGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGC GGCCGCCACTAGTGGCCAGGCCGGCCAGGAGAGCAAGTACG GCCCTCCCTGCCCCCCTTGCCCTTTTTGGGTGCTGGTGGTGGT TGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCT GCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC CCACTAGTACCCGCAAGCATTACCAGCCCTATGCCCCACCAC

[0395]

[0396] GCGACTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGAAA Attorney Docket No: 046483 -7482WO 1(04099)

[0397] CTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAA ACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA AGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGC AGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCA GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACG ATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACA ATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAG ATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG ATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT ACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA MSLN VHH16.28BBz CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGCC CAR DNA TGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCAC CTTCAGTATGTCTACCATGAACTGGGTCCGCCAAACCCCAGG AAAGGGGCTCGAGTGGGTCTCCGGTATTAACAATAGTGGTG GATTTACAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCA TCTCCAGAGACAACGCCAAGAATATGCTGTATCTGCAAATG AATAGCCTGAAACCTGAGGACACGGCTGTGTATTACTGTACA AGATTTGCTAGTTCGTTATCTCCACCCGTGAGCCAGGGGACC CAGGTCACCGTCTCCTCAGCGGCCGCCACTAGTGGCCAGGCC GGCCAGGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCT TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTAT AGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGG AGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACAT GACTCCCCGCCGCCCCGGGCCCACTAGTACCCGCAAGCATTA CCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAAC CATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGC TGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGA ACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGT ACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTA GGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGG CCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAAC CCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCC GGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT ACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC CCTGCCCCCTCGCTAA MSLN VHH18.28BBz CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGC CAR DNA TGGGGGCTCTCTACGACTCTCCTGTGCAGCCTCTGAACGCAC CTTCAGTACTTATGCCATGGCCTGGTTCCGCCAGGCTCCAGG GAAGGAGCGTGAATTTGTAGCAGCTATTGAGTATAGTGTTGC AGTGACAACGTATGCAGACTCCGTCAAGGGCCGATTCACCA TCTCCAGAGACATCGCCCAGAACACGGTGTATCTGCAAATG AACAGCCTGAAACCTGACGACACGGCCGTTTATTTTTGTGCA GCACGTCCTTCGCCTATGGCGACTTCATCCGCTCAAACCTAT GAGTATTGGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGC GGCCGCCACTAGTGGCCAGGCCGGCCAGGAGAGCAAGTACG GCCCTCCCTGCCCCCCTTGCCCTTTTTGGGTGCTGGTGGTGGT TGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC

[0398]

[0399] CTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCT Attorney Docket No: 046483 -7482WO 1(04099)

[0400] GCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC CCACTAGTACCCGCAAGCATTACCAGCCCTATGCCCCACCAC GCGACTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGAAA CTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAA ACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA AGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGC AGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCA GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACG ATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACA ATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAG ATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG ATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT ACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA MSLN VHH61.28BBz CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGT CAR DNA TGGGGACTCTCTTAGACTCTCCTGTGCAGCCTCTGAACGCAT CTTCAGTACGTATGCCCAGGGCTGGTTCCGCCAGGCTCCAGG GAAGGAGCGTGAATTTGTAGCAAGCATTGGCTATAGTGCGA CTGACACAAGATATGCAGACTCCGTGAAGGGCCGATTCACC ATCTCCAGAGACAGCGCCAAGAACACGATCGATCTACTTAT GAACAGCCTGAAACCTGACGACACGGCCGTTTATTACTGTGC AGGACGTCCTTCGCCAGTACCGACTTCATCCGCTCAACTCTA TGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGC GGCCGCCACTAGTGGCCAGGCCGGCCAGGAGAGCAAGTACG GCCCTCCCTGCCCCCCTTGCCCTTTTTGGGTGCTGGTGGTGGT TGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCT GCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC CCACTAGTACCCGCAAGCATTACCAGCCCTATGCCCCACCAC GCGACTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGAAA CTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAA ACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA AGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGC AGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCA GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACG ATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACA ATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAG ATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG ATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT ACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA MSLN VHH67.28BBz CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGC CAR DNA TGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGAACGCAC CTTCAGTACGTATGCCATGGGCTGGTTCCGCCAGGCTCCAGG GAAGGAGCGTGAATTTGTAGCGGCTATTGGCTACAGTCATGC TATGACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCAT TTCCCGAGACAACGCCAAGAACACGGTGTATCTGCAAATGA ACAGCCTGAAGTCATACGACACGGCCGTTTATTTTTGTGCAG CACGTCCTTCGCCTATGGCGACTTTATCCGCTCAAACCTATG ACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCG

[0401]

[0402] GCCGCCACTAGTGGCCAGGCCGGCCAGGAGAGCAAGTACGG Attorney Docket No: 046483 -7482WO 1(04099)

[0403] CCCTCCCTGCCCCCCTTGCCCTTTTTGGGTGCTGGTGGTGGTT GGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCC TTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTG CACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCC ACTAGTACCCGCAAGCATTACCAGCCCTATGCCCCACCACGC GACTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGAAACT CCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAAC TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAG AAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGC TCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGAT GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATG AACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATG GCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA MSLN VHH68.28BBz CAGGTGCAGCTGCAGGAGTCTGGGGGAGGAGTGGTGCAGGC CAR DNA TGGGGGCTCTCTGAGACTCTCCTGTGTAGGCTCTGGACGCAT CTTCGGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCTGG AAAGGAGCGTGAGTTTGTTGCAGCAATTGCGTATAACTTCGA CACAAAGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTC CAGAGACAACGCCAGGAACACGCTGTATCTGCAAATGAACA ACCTGAAACCTGAAGACACGGCGGTTTATTACTGTGCACAA AGATCAGGGCCCGGATTTAGTGATAACCTTCGTCTGTATGAC TACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGC CGCCACTAGTGGCCAGGCCGGCCAGGAGAGCAAGTACGGCC CTCCCTGCCCCCCTTGCCCTTTTTGGGTGCTGGTGGTGGTTGG TGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTT TATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCA CAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCAC TAGTACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGA CTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGAAACTCC TGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTA CTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAA GAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGA GCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTC TATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGT TTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAA CTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGG GATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGC CTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC GCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA VHHB30-30BBz CAR CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGC DNA - (no signal TGGGGGGTCTCTAAAACTCTCCTGTGCAGCCTCTGGAAGCAT peptide) CCACAGTATCAATGCCATGGGCTGGTACCGCCAGACTCCAG GGAACCAGCGCGAGTTGGTCGCAACTATTACTAATGGTGTTA ATGGTGTCACCACAAACTATGCAAACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTG

[0404]

[0405] CAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTA Attorney Docket No: 046483 -7482WO 1(04099)

[0406] CTGTACACCAGACCCCCTGCGGTACCTGTTTGATTCCTGGGG CCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCCACTA GTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCC CTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTG GGTGAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAA GATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGG ATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCC CCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAG ACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGG AAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCG AGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGT CTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG CAGGCCCTGCCCCCTCGCTAA VHHB30-28BBz CAR CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGC DNA (3rd gen, no TGGGGGGTCTCTAAAACTCTCCTGTGCAGCCTCTGGAAGCAT signal peptide) CCACAGTATCAATGCCATGGGCTGGTACCGCCAGACTCCAG GGAACCAGCGCGAGTTGGTCGCAACTATTACTAATGGTGTTA ATGGTGTCACCACAAACTATGCAAACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTA CTGTACACCAGACCCCCTGCGGTACCTGTTTGATTCCTGGGG CCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCCACTA GTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCC CTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTG GGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACA TGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATT ACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCT CCGCTAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTC AAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGA AGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG GATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCC CCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCT CAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGA GACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC GAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGG TCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAGGCCCTGCCCCCTCGCTAA VHHB30-30BBz CAR ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTG DNA CTGCTCCACGCCGCCAGGCCGGGATCCGCGGCCCAGGCGGC CCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGG CTGGGGGGTCTCTAAAACTCTCCTGTGCAGCCTCTGGAAGCA TCCACAGTATCAATGCCATGGGCTGGTACCGCCAGACTCCAG GGAACCAGCGCGAGTTGGTCGCAACTATTACTAATGGTGTTA

[0407]

[0408] ATGGTGTCACCACAAACTATGCAAACTCCGTGAAGGGCCGA Attorney Docket No: 046483 -7482WO 1(04099)

[0409] TTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTA CTGTACACCAGACCCCCTGCGGTACCTGTTTGATTCCTGGGG CCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCCACTA GTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCC CTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTG GGTGAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAA GATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGG ATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCC CCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAG ACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGG AAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCG AGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGT CTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG CAGGCCCTGCCCCCTCGCTAA VHHB30-28BBz CAR ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTG DNA (3rd gen) CTGCTCCACGCCGCCAGGCCGGGATCCGCGGCCCAGGCGGC CCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGG CTGGGGGGTCTCTAAAACTCTCCTGTGCAGCCTCTGGAAGCA TCCACAGTATCAATGCCATGGGCTGGTACCGCCAGACTCCAG GGAACCAGCGCGAGTTGGTCGCAACTATTACTAATGGTGTTA ATGGTGTCACCACAAACTATGCAAACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTA CTGTACACCAGACCCCCTGCGGTACCTGTTTGATTCCTGGGG CCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCCACTA GTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCC CTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTG GGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACA TGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATT ACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCT CCGCTAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTC AAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGA AGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG GATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCC CCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCT CAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGA GACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC GAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGG TCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAGGCCCTGCCCCCTCGCTAA CEACAM5 MALPVTALLLPLALLLHAARPGSAAQAAQVQLQESGGGLVQA VHHB30.28BBz CAR GGSLKLSCAASGSIHSINAMGWYRQTPGNQRELVATITNGVNG

[0410]

[0411] protein VTTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTP Attorney Docket No: 046483 -7482WO 1(04099)

[0412] DPLRYLFDSWGQGTQVTVSSAAATSSGESKYGPPCPPCPDIFWV LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSASKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR CEACAM5 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTG VHHB30.28BBz CAR CTGCTCCACGCCGCCAGGCCGGGATCCGCGGCCCAGGCGGC DNA CCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGG CTGGGGGGTCTCTAAAACTCTCCTGTGCAGCCTCTGGAAGCA TCCACAGTATCAATGCCATGGGCTGGTACCGCCAGACTCCAG GGAACCAGCGCGAGTTGGTCGCAACTATTACTAATGGTGTTA ATGGTGTCACCACAAACTATGCAAACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTA CTGTACACCAGACCCCCTGCGGTACCTGTTTGATTCCTGGGG CCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCCACTA GTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCC CTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGG CTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTG GGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACA TGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATT ACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCT CCGCTAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTC AAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGA AGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG GATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCC CCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCT CAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGA GACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC GAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGG TCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAGGCCCTGCCCCCTCGCTAA MSLN QVQLQESGGGSVQPGGSLRLSCAASGFTFSMSAMNWVRQTAG VHH1.28BBz / CEACA KGLEWVSGITNSGGFTSYADSVKGRFTISRDNAKNTLYLQMNS M5 VHHB30. BB Split LKPEDTAVYYCTRFASSLSPPVSQGTQVTVSSGQAGQESKYGPP Bi-CAR protein CPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY MNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPRIDRRKRGSGATNFSLLKQAGDVEENPGP GSGMALPVTALLLPLALLLHAARPGSQVQLQESGGGLVQAGGS LKLSCAASGSIHSINAMGWYRQTPGNQRELVATITNGVNGVTT NYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTPDPL RYLFDSWGQGTQVTVSSSGESKYGPPCPPCPFWVLVVVGGVLA CYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCS

[0413]

[0414] CRFPEEEEGGCEL Attorney Docket No: 046483 -7482WO 1(04099)

[0415] MSLN- QVQLQESGGGLVQTGGSLRLSCAASRSIFSTYAMGWFRQAPGK VHH12.28BBz / CEAC EREFVAAIGYSHGNTDYVDSVKGRFTISRDNAKNTMYLQMNSL AM5 RPEDTAVYYCAAYTGPRPPRQAPLYDYWGQGTQVTVSSGQAG VHHB30. BB / Split BiQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR CAR protein SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPRIDRRKRGSGATNFSLLKQAG DVEENPGPGSGMALPVTALLLPLALLLHAARPGSQVQLQESGG GLVQAGGSLKLSCAASGSIHSINAMGWYRQTPGNQRELVATIT NGVNGVTTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCTPDPLRYLFDSWGQGTQVTVSSSGESKYGPPCPPCPFWV LVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCEL CEA QVQLQESGGGLVQAGGSLKLSCAASGSIHSINAMGWYRQTPGN VHHB30.28BBz / MSL QRELVATITNGVNGVTTNYANSVKGRFTISRDNAKNTVYLQM NVHHl. BB / Split BiNSLKPEDTAVYYCTPDPLRYLFDSWGQGTQVTVSSGQAGQESK CAR protein YGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPRIDRRKRGSGATNFSLLKQAGDVE ENPGPGSGMALPVTALLLPLALLLHAARPGSQVQLQESGGGSV QPGGSLRLSCAASGFTFSMSAMNWVRQTAGKGLEWVSGITNS GGFTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCT RFASSLSPPVSQGTQVTVSSSGESKYGPPCPPCPFWVLVVVGGV LACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCEL CEA QVQLQESGGGLVQAGGSLKLSCAASGSIHSINAMGWYRQTPGN VHHB30.28BBz / MSL QRELVATITNGVNGVTTNYANSVKGRFTISRDNAKNTVYLQM N VHH12. BB / Split BiNSLKPEDTAVYYCTPDPLRYLFDSWGQGTQVTVSSGQAGQESK CAR protein YGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPRIDRRKRGSGATNFSLLKQAGDVE ENPGPGSGMALPVTALLLPLALLLHAARPGSQVQLQESGGGLV QTGGSLRLSCAASRSIFSTYAMGWFRQAPGKEREFVAAIGYSH GNTDYVDSVKGRFTISRDNAKNTMYLQMNSLRPEDTAVYYCA AYTGPRPPRQAPLYDYWGQGTQVTVSSSGESKYGPPCPPCPFW VLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCEL MSLN- QVQLQESGGGSVQPGGSLRLSCAASGFTFSMSAMNWVRQTAG VHH1 / CEACAM5- KGLEWVSGITNSGGFTSYADSVKGRFTISRDNAKNTLYLQMNS VHHB30.28BBz / Tande LKPEDTAVYYCTRFASSLSPPVSQGTQVTVSSHMGSGSGSGSGS m Bi-CAR protein GSGSGSASQVQLQESGGGLVQAGGSLKLSCAASGSIHSINAMG WYRQTPGNQRELVATITNGVNGVTTNYANSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCTPDPLRYLFDSWGQGTQVTVSSS GGQAGQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIF WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR

[0416]

[0417] SKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR Attorney Docket No: 046483 -7482WO 1(04099)

[0418] VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR MSLN- QVQLQESGGGLVQTGGSLRLSCAASRSIFSTYAMGWFRQAPGK VHH12 / CEACAM5- EREFVAAIGYSHGNTDYVDSVKGRFTISRDNAKNTMYLQMNSL VHHB30.28BBz / Tande RPEDTAVYYCAAYTGPRPPRQAPLYDYWGQGTQVTVSSHMGS m Bi-CAR Protein GSGSGSGSGSGSGSASQVQLQESGGGLVQAGGSLKLSCAASGSI HSINAMGWYRQTPGNQRELVATITNGVNGVTTNYANSVKGRF TISRDNAKNTVYLQMNSLKPEDTAVYYCTPDPLRYLFDSWGQG TQVTVSSSGGQAGQESKYGPPCPPCPFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CEACAM5- QVQLQESGGGLVQAGGSLKLSCAASGSIHSINAMGWYRQTPGN VHHB30 / MSLN- QRELVATITNGVNGVTTNYANSVKGRFTISRDNAKNTVYLQM VHH1.28BBz / Tandem NSLKPEDTAVYYCTPDPLRYLFDSWGQGTQVTVSSHMGSGSGS Bi-CAR protein GSGSGSGSGSASQVQLQESGGGSVQPGGSLRLSCAASGFTFSMS AMNWVRQTAGKGLEWVSGITNSGGFTSYADSVKGRFTISRDN AKNTLYLQMNSLKPEDTAVYYCTRFASSLSPPVSQGTQVTVSSS GGQAGQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIF WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR SKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR CEACAM5- QVQLQESGGGLVQAGGSLKLSCAASGSIHSINAMGWYRQTPGN VHHB30 / MSLN- QRELVATITNGVNGVTTNYANSVKGRFTISRDNAKNTVYLQM VHH12.28BBz / Tande NSLKPEDTAVYYCTPDPLRYLFDSWGQGTQVTVSSHMGSGSGS m Bi-CAR protein GSGSGSGSGSASQVQLQESGGGLVQTGGSLRLSCAASRSIFSTY AMGWFRQAPGKEREFVAAIGYSHGNTDYVDSVKGRFTISRDN AKNTMYLQMNSLRPEDTAVYYCAAYTGPRPPRQAPLYDYWG QGTQVTVSSSGGQAGQESKYGPPCPPCPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR MSLN CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGCC VHH1.28BBz / CEA TGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCAC VHHB30. BB / Split Bi- CTTCAGTATGTCTGCCATGAACTGGGTCCGCCAAACTGCAGG CAR DNA CAAGGGGCTCGAGTGGGTCTCCGGTATTACTAATAGTGGTGG ATTTACAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCAT CTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGA ATAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTACA AGATTTGCTAGTTCGTTATCTCCACCCGTGAGCCAGGGGACC CAGGTCACCGTCTCCTCAGGCCAGGCCGGCCAGGAATCAAA ATATGGTCCCCCGTGCCCACCTTGCCCGTTTTGGGTCCTCGTC

[0419]

[0420] GTAGTTGGCGGCGTTCTGGCTTGTTACAGCCTTCTCGTGACT Attorney Docket No: 046483 -7482WO 1(04099)

[0421] GTCGCGTTTATTATTTTCTGGGTACGGTCAAAAAGGTCCAGA TTGCTGCATTCAGATTACATGAACATGACACCTCGCCGGCCC GGGCCCACGCGCAAGCATTATCAGCCCTACGCGCCTCCGAG AGACTTCGCTGCTTATAGGAGTAGGGTTAAGTTTTCACGCTC CGCAGATGCCCCAGCATACCAGCAGGGTCAGAATCAGCTGT ATAATGAGTTGAACTTGGGTAGACGCGAGGAATATGACGTG CTGGATAAACGACGCGGCCGCGACCCTGAGATGGGGGGGAA ACCACGGAGAAAGAACCCCCAGGAGGGCTTGTATAACGAAC TGCAAAAAGACAAAATGGCCGAAGCGTACTCCGAGATCGGG ATGAAAGGAGAGCGGCGACGAGGAAAGGGTCATGATGGTCT TTATCAGGGTCTGTCTACTGCAACAAAAGACACTTACGACGC GCTTCACATGCAGGCCTTGCCCCCGCGCATTGACAGGCGCAA ACGCGGGTCTGGCGCTACAAACTTCTCCCTGCTTAAACAGGC AGGGGACGTTGAGGAAAACCCAGGTCCTGGGTCTGGAATGG CGTTGCCAGTGACCGCGCTTTTGCTCCCATTGGCACTGCTTTT GCACGCAGCCCGGCCAGGATCCCAGGTACAATTGCAAGAGA GTGGAGGTGGGCTGGTTCAGGCAGGGGGCTCACTTAAGCTT AGCTGTGCGGCATCTGGCAGCATACATTCTATAAATGCCATG GGCTGGTACCGGCAGACTCCGGGCAATCAGCGGGAGTTGGT AGCTACAATTACTAACGGGGTGAATGGTGTGACCACTAACT ACGCAAACTCAGTTAAAGGACGGTTTACTATCTCACGGGAC AACGCGAAAAACACCGTCTACCTGCAGATGAACAGCCTCAA ACCTGAAGACACAGCAGTTTATTATTGCACACCAGATCCTCT CCGCTATCTCTTTGACTCTTGGGGTCAGGGTACCCAGGTAAC CGTCTCTTCCTCCGGAGAAAGTAAATACGGTCCCCCGTGTCC TCCATGCCCTTTCTGGGTTTTGGTCGTTGTCGGCGGCGTTTTG GCGTGTTACTCTCTTTTGGTCACGGTGGCTTTCATTATTTTTT GGGTTAAAAGAGGTCGGAAGAAATTGTTGTATATCTTTAAAC AACCGTTCATGAGGCCTGTGCAAACAACTCAGGAGGAGGAT GGCTGCAGCTGTAGATTTCCAGAAGAAGAAGAGGGTGGATG TGAATTGTAA MSLN- CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGAC VHH12.28BBz / CEA TGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTAGAAGTAT VHHB30. BB / Split BiTTTCAGTACATATGCCATGGGCTGGTTCCGCCAGGCTCCAGG CAR DNA GAAGGAGCGTGAGTTTGTAGCGGCTATTGGTTATAGTCATGG AAACACAGACTATGTAGACTCCGTGAAGGGCCGATTCACCA TCTCCAGAGACAACGCCAAGAACACGATGTATCTGCAAATG AACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCA GCATACACAGGACCTCGGCCACCACGGCAGGCGCCCTTATA TGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAG GCCAGGCCGGCCAGGAATCAAAATATGGTCCCCCGTGCCCA CCTTGCCCGTTTTGGGTCCTCGTCGTAGTTGGCGGCGTTCTGG CTTGTTACAGCCTTCTCGTGACTGTCGCGTTTATTATTTTCTG GGTACGGTCAAAAAGGTCCAGATTGCTGCATTCAGATTACAT GAACATGACACCTCGCCGGCCCGGGCCCACGCGCAAGCATT ATCAGCCCTACGCGCCTCCGAGAGACTTCGCTGCTTATAGGA GTAGGGTTAAGTTTTCACGCTCCGCAGATGCCCCAGCATACC AGCAGGGTCAGAATCAGCTGTATAATGAGTTGAACTTGGGT AGACGCGAGGAATATGACGTGCTGGATAAACGACGCGGCCG CGACCCTGAGATGGGGGGGAAACCACGGAGAAAGAACCCCC

[0422]

[0423] AGGAGGGCTTGTATAACGAACTGCAAAAAGACAAAATGGCC Attorney Docket No: 046483 -7482WO 1(04099)

[0424] GAAGCGTACTCCGAGATCGGGATGAAAGGAGAGCGGCGACG AGGAAAGGGTCATGATGGTCTTTATCAGGGTCTGTCTACTGC AACAAAAGACACTTACGACGCGCTTCACATGCAGGCCTTGC CCCCGCGCATTGACAGGCGCAAACGCGGGTCTGGCGCTACA AACTTCTCCCTGCTTAAACAGGCAGGGGACGTTGAGGAAAA CCCAGGTCCTGGGTCTGGAATGGCGTTGCCAGTGACCGCGCT TTTGCTCCCATTGGCACTGCTTTTGCACGCAGCCCGGCCAGG ATCCCAGGTACAATTGCAAGAGAGTGGAGGTGGGCTGGTTC AGGCAGGGGGCTCACTTAAGCTTAGCTGTGCGGCATCTGGC AGCATACATTCTATAAATGCCATGGGCTGGTACCGGCAGACT CCGGGCAATCAGCGGGAGTTGGTAGCTACAATTACTAACGG GGTGAATGGTGTGACCACTAACTACGCAAACTCAGTTAAAG GACGGTTTACTATCTCACGGGACAACGCGAAAAACACCGTC TACCTGCAGATGAACAGCCTCAAACCTGAAGACACAGCAGT TTATTATTGCACACCAGATCCTCTCCGCTATCTCTTTGACTCT TGGGGTCAGGGTACCCAGGTAACCGTCTCTTCCTCCGGAGAA AGTAAATACGGTCCCCCGTGTCCTCCATGCCCTTTCTGGGTTT TGGTCGTTGTCGGCGGCGTTTTGGCGTGTTACTCTCTTTTGGT CACGGTGGCTTTCATTATTTTTTGGGTTAAAAGAGGTCGGAA GAAATTGTTGTATATCTTTAAACAACCGTTCATGAGGCCTGT GCAAACAACTCAGGAGGAGGATGGCTGCAGCTGTAGATTTC CAGAAGAAGAAGAGGGTGGATGTGAATTGTAA CEA CAGGTACAACTGCAGGAGAGCGGCGGCGGTCTTGTCCAAGC VHHB30.28BBz / MSL AGGTGGCTCACTCAAACTCTCTTGTGCAGCCAGTGGATCAAT N VHHl. BB / Split BiACATTCTATTAACGCTATGGGGTGGTATAGGCAAACACCCGG CAR DNA CAATCAGCGGGAACTGGTAGCCACAATTACAAACGGTGTAA ATGGAGTTACCACGAATTACGCTAATTCCGTCAAGGGCCGAT TTACCATTTCACGGGACAATGCAAAGAATACCGTCTATTTGC AGATGAACAGCCTCAAGCCGGAAGACACCGCGGTTTATTAT TGCACCCCCGATCCGCTTAGGTACCTTTTTGATTCTTGGGGCC AGGGTACCCAGGTTACTGTATCATCCGGCCAGGCCGGCCAG GAGTCTAAATACGGCCCGCCTTGTCCGCCATGTCCATTTTGG GTGTTGGTGGTCGTAGGCGGAGTCCTTGCGTGTTACTCCCTG TTGGTAACCGTTGCTTTTATAATTTTTTGGGTGCGGTCTAAGC GAAGTCGCTTGCTCCACAGTGATTATATGAACATGACGCCGC GCAGACCCGGGCCTACCAGAAAGCATTACCAGCCGTATGCG CCGCCTAGGGACTTTGCTGCATATAGATCCAGAGTGAAGTTT TCCCGATCAGCAGACGCCCCAGCGTATCAACAGGGACAAAA TCAGTTGTACAACGAGCTGAATCTCGGTCGCAGAGAAGAGT ATGATGTGCTCGACAAAAGACGAGGGCGGGACCCTGAAATG GGCGGGAAACCTAGAAGGAAAAACCCACAAGAAGGGCTGT ACAATGAGCTTCAAAAAGATAAAATGGCAGAGGCATACAGT GAAATAGGCATGAAAGGGGAGCGCCGGCGAGGAAAAGGCC ACGACGGACTGTATCAAGGTCTTAGTACTGCTACGAAGGAC ACCTACGACGCGTTGCATATGCAAGCATTGCCCCCCAGAATA GACAGGAGAAAACGCGGAAGTGGGGCAACAAACTTCTCTCT TCTTAAGCAAGCGGGAGACGTGGAAGAGAACCCGGGACCAG GATCTGGGATGGCTTTGCCCGTTACTGCACTTCTCCTTCCACT CGCACTTCTCTTGCACGCAGCCCGGCCGGGATCCCAGGTGCA GCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGCCTGGGGGGT

[0425]

[0426] CTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAT Attorney Docket No: 046483 -7482WO 1(04099)

[0427] GTCTGCCATGAACTGGGTCCGCCAAACTGCAGGCAAGGGGC TCGAGTGGGTCTCCGGTATTACTAATAGTGGTGGATTTACAA GCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAG ACAACGCCAAGAACACGCTGTATCTGCAAATGAATAGCCTG AAACCTGAGGACACGGCCGTGTATTACTGTACAAGATTTGCT AGTTCGTTATCTCCACCCGTGAGCCAGGGGACCCAGGTCACC GTCTCCTCATCCGGAGAATCAAAATATGGTCCCCCATGTCCA CCTTGTCCATTTTGGGTTCTTGTTGTAGTTGGAGGCGTCTTGG CGTGTTATAGCTTGCTCGTCACTGTTGCATTTATAATATTCTG GGTAAAGCGGGGTAGGAAAAAACTGCTCTATATCTTCAAGC AGCCTTTTATGAGGCCGGTTCAAACCACCCAAGAGGAGGAT GGATGTAGCTGTCGATTTCCAGAAGAGGAAGAGGGCGGTTG CGAACTCTGA CEA CAGGTACAACTGCAGGAGAGCGGCGGCGGTCTTGTCCAAGC VHHB30.28BBz / MSL AGGTGGCTCACTCAAACTCTCTTGTGCAGCCAGTGGATCAAT N VHH 12. BB / Split BiACATTCTATTAACGCTATGGGGTGGTATAGGCAAACACCCGG CAR DNA CAATCAGCGGGAACTGGTAGCCACAATTACAAACGGTGTAA ATGGAGTTACCACGAATTACGCTAATTCCGTCAAGGGCCGAT TTACCATTTCACGGGACAATGCAAAGAATACCGTCTATTTGC AGATGAACAGCCTCAAGCCGGAAGACACCGCGGTTTATTAT TGCACCCCCGATCCGCTTAGGTACCTTTTTGATTCTTGGGGCC AGGGTACCCAGGTTACTGTATCATCCGGCCAGGCCGGCCAG GAGTCTAAATACGGCCCGCCTTGTCCGCCATGTCCATTTTGG GTGTTGGTGGTCGTAGGCGGAGTCCTTGCGTGTTACTCCCTG TTGGTAACCGTTGCTTTTATAATTTTTTGGGTGCGGTCTAAGC GAAGTCGCTTGCTCCACAGTGATTATATGAACATGACGCCGC GCAGACCCGGGCCTACCAGAAAGCATTACCAGCCGTATGCG CCGCCTAGGGACTTTGCTGCATATAGATCCAGAGTGAAGTTT TCCCGATCAGCAGACGCCCCAGCGTATCAACAGGGACAAAA TCAGTTGTACAACGAGCTGAATCTCGGTCGCAGAGAAGAGT ATGATGTGCTCGACAAAAGACGAGGGCGGGACCCTGAAATG GGCGGGAAACCTAGAAGGAAAAACCCACAAGAAGGGCTGT ACAATGAGCTTCAAAAAGATAAAATGGCAGAGGCATACAGT GAAATAGGCATGAAAGGGGAGCGCCGGCGAGGAAAAGGCC ACGACGGACTGTATCAAGGTCTTAGTACTGCTACGAAGGAC ACCTACGACGCGTTGCATATGCAAGCATTGCCCCCCAGAATA GACAGGAGAAAACGCGGAAGTGGGGCAACAAACTTCTCTCT TCTTAAGCAAGCGGGAGACGTGGAAGAGAACCCGGGACCAG GATCTGGGATGGCTTTGCCCGTTACTGCACTTCTCCTTCCACT CGCACTTCTCTTGCACGCAGCCCGGCCGGGATCCCAGGTGCA GCTGCAGGAGTCTGGAGGAGGATTGGTGCAGACTGGGGGCT CTCTGAGACTCTCCTGTGCAGCCTCTAGAAGTATTTTCAGTA CATATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAG CGTGAGTTTGTAGCGGCTATTGGTTATAGTCATGGAAACACA GACTATGTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGA GACAACGCCAAGAACACGATGTATCTGCAAATGAACAGCCT GAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCATACAC AGGACCTCGGCCACCACGGCAGGCGCCCTTATATGACTACTG GGGCCAGGGGACCCAGGTCACCGTCTCCTCATCCGGAGAAT CAAAATATGGTCCCCCATGTCCACCTTGTCCATTTTGGGTTCT

[0428]

[0429] TGTTGTAGTTGGAGGCGTCTTGGCGTGTTATAGCTTGCTCGT Attorney Docket No: 046483 -7482WO 1(04099)

[0430] CACTGTTGCATTTATAATATTCTGGGTAAAGCGGGGTAGGAA AAAACTGCTCTATATCTTCAAGCAGCCTTTTATGAGGCCGGT TCAAACCACCCAAGAGGAGGATGGATGTAGCTGTCGATTTC CAGAAGAGGAAGAGGGCGGTTGCGAACTCTGA MSLN- CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGCC VHH1 / CEACAM5- TGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCAC VHHB30.28BBz / Tande CTTCAGTATGTCTGCCATGAACTGGGTCCGCCAAACTGCAGG m Bi-CAR DNA CAAGGGGCTCGAGTGGGTCTCCGGTATTACTAATAGTGGTGG ATTTACAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCAT CTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGA ATAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTACA AGATTTGCTAGTTCGTTATCTCCACCCGTGAGCCAGGGGACC CAGGTCACCGTCTCCTCACATATGGGCTCCGGCAGTGGGTCA GGATCAGGTTCAGGTAGCGGGTCAGGTTCCGCTAGCCAGGT GCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGG GGTCTCTAAAACTCTCCTGTGCAGCCTCTGGAAGCATCCACA GTATCAATGCCATGGGCTGGTACCGCCAGACTCCAGGGAAC CAGCGCGAGTTGGTCGCAACTATTACTAATGGTGTTAATGGT GTCACCACAAACTATGCAAACTCCGTGAAGGGCCGATTCAC CATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAA TGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTACTGTA CACCAGACCCCCTGCGGTACCTGTTTGATTCCTGGGGCCAGG GGACCCAGGTCACCGTCTCCTCATCCGGAGGCCAGGCCGGC CAGGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTTTT TGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGC TTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGT AAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGAC TCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTA TGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAACGGGG CAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAG ACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTG AAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGG CCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCT GAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG GCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCC TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTC GCTAA MSLN- CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGAC VHH12 / CEACAM5- TGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCAGAAGTAT VHHB30.28BBz / Tande TTTCAGTACGTATGCCATGGGCTGGTTCCGCCAGGCTCCAGG m Bi-CAR DNA GAAGGAGCGTGAGTTTGTAGCGGCTATTGGTTATAGTCATGG AAACACAGACTATGTAGACTCCGTGAAGGGCCGATTCACCA TCTCCAGAGACAACGCCAAGAACACGATGTATCTGCAAATG AACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCA GCATACACAGGACCTCGGCCACCACGGCAGGCGCCCTTATA TGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCACA

[0431]

[0432] TATGGGCTCCGGCAGTGGGTCAGGATCAGGTTCAGGTAGCG Attorney Docket No: 046483 -7482WO 1(04099)

[0433] GGTCAGGTTCCGCTAGCCAGGTGCAGCTGCAGGAGTCTGGG GGAGGCTTGGTGCAGGCTGGGGGGTCTCTAAAACTCTCCTGT GCAGCCTCTGGAAGCATCCACAGTATCAATGCCATGGGCTG GTACCGCCAGACTCCAGGGAACCAGCGCGAGTTGGTCGCAA CTATTACTAATGGTGTTAATGGTGTCACCACAAACTATGCAA ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGA GGACACAGCCGTCTATTACTGTACACCAGACCCCCTGCGGTA CCTGTTTGATTCCTGGGGCCAGGGGACCCAGGTCACCGTCTC CTCATCCGGAGGCCAGGCCGGCCAGGAGAGCAAGTACGGCC CTCCCTGCCCCCCTTGCCCTTTTTGGGTGCTGGTGGTGGTTGG TGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTT TATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCA CAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCAC CCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGC AGCCTATCGCTCCAAACGGGGCAGAAAGAAACTCCTGTATA TATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAG AGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAA GGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACG AGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGA GAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAA AGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACC AGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTT CACATGCAGGCCCTGCCCCCTCGCTAA CEACAM5- CAGGTGCAATTGCAAGAGAGCGGAGGCGGGCTTGTCCAGGC VHHB30 / MSLN- CGGTGGGTCTCTCAAACTTTCTTGCGCCGCGTCTGGTAGTAT VHH1.28BBz / Tandem ACATTCAATCAACGCAATGGGATGGTATCGGCAAACACCCG Bi-CAR DNA GCAACCAACGGGAGCTCGTAGCGACCATTACAAATGGTGTC AATGGCGTGACCACCAACTACGCGAACTCTGTCAAAGGACG CTTTACTATATCTAGGGATAACGCGAAGAACACCGTTTACCT GCAGATGAATAGTCTTAAGCCTGAAGATACAGCGGTATATT ACTGCACACCTGATCCGCTGCGCTACTTGTTTGACAGCTGGG GCCAAGGGACGCAAGTCACAGTCAGCTCACATATGGGCTCC GGCAGTGGGTCAGGATCAGGTTCAGGTAGCGGGTCAGGTTC CGCTAGCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGG TGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTG GATTCACCTTCAGTATGTCTGCCATGAACTGGGTCCGCCAAA CTGCAGGCAAGGGGCTCGAGTGGGTCTCCGGTATTACTAATA GTGGTGGATTTACAAGCTATGCAGACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTG CAAATGAATAGCCTGAAACCTGAGGACACGGCCGTGTATTA CTGTACAAGATTTGCTAGTTCGTTATCTCCACCCGTGAGCCA GGGGACCCAGGTCACCGTCTCCTCATCCGGAGGCCAGGCCG GCCAGGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTT TTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATA GCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGA GTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATG

[0434]

[0435] ACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCC Attorney Docket No: 046483 -7482WO 1(04099)

[0436] TATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAACGG GGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATG AGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAG TGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAG GGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAG AGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACC CTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGA AGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGG CCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGC AAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCAC CAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCC TCGCTAA CEACAM5- CAGGTGCAATTGCAAGAGAGCGGAGGCGGGCTTGTCCAGGC VHHB30 / MSLN- CGGTGGGTCTCTCAAACTTTCTTGCGCCGCGTCTGGTAGTAT VHH12.28BBz / Tande ACATTCAATCAACGCAATGGGATGGTATCGGCAAACACCCG m Bi-CAR DNA GCAACCAACGGGAGCTCGTAGCGACCATTACAAATGGTGTC AATGGCGTGACCACCAACTACGCGAACTCTGTCAAAGGACG CTTTACTATATCTAGGGATAACGCGAAGAACACCGTTTACCT GCAGATGAATAGTCTTAAGCCTGAAGATACAGCGGTATATT ACTGCACACCTGATCCGCTGCGCTACTTGTTTGACAGCTGGG GCCAAGGGACGCAAGTCACAGTCAGCTCACATATGGGCTCC GGCAGTGGGTCAGGATCAGGTTCAGGTAGCGGGTCAGGTTC CGCTAGCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGG TGCAGACTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTA GAAGTATTTTCAGTACATATGCCATGGGCTGGTTCCGCCAGG CTCCAGGGAAGGAGCGTGAGTTTGTAGCGGCTATTGGTTATA GTCATGGAAACACAGACTATGTAGACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACACGATGTATCTG CAAATGAACAGCCTGAGACCTGAGGACACGGCCGTTTATTA CTGTGCAGCATACACAGGACCTCGGCCACCACGGCAGGCGC CCTTATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCT CCTCATCCGGAGGCCAGGCCGGCCAGGAGAGCAAGTACGGC CCTCCCTGCCCCCCTTGCCCTTTTTGGGTGCTGGTGGTGGTTG GTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCT TTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGC ACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCC ACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTC GCAGCCTATCGCTCCAAACGGGGCAGAAAGAAACTCCTGTA TATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCA AGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAG AAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCA GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGG ACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCC GAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGC AGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATG AAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC

[0437]

[0438] TTCACATGCAGGCCCTGCCCCCTCGCTAA Attorney Docket No: 046483 -7482WO 1(04099) pTRPE-VHHB3O- GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGT 30BBz AAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACA CTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGA AAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCA GTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTAC TTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTT TGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG AACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGAC ACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACT ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATT AATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTC TGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAAT CTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA CGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAG ATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTG TCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAA CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTG ATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCC ACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAG CGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAG GCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCG CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAG GGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGG AACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATG GAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTT TTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCT GATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTC TCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACA GGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATT AATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACAC TTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGA TAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCC AAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGA GCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTT GCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGA GAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTG GTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGA

[0439]

[0440] Attorney Docket No: 046483 -7482WO 1(04099)

[0441] CATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGAT ATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTC TGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTA GGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTG CTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAAC TAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCT AGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAA ACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAG CGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACG CCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGG GTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGC GATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAA ATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAG AACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAG GCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAG ACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGC AACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACA CCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAA AAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGA CCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATT ATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAG CACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAA AAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGG AGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGA CGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGC AGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTG TTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAG AATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCC TGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTG CTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAAC AGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAA ATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAA TCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGA ATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAAC AAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGG AGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATA GTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAG ACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGG AATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCC ATTCGATTAGTGAACGGATCTCGACGGTATCGATTAGACTGT AGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGG AAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATAT AGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACA GCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAA AACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTA CAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAA TTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAA TCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAG AGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAG TATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGG

[0442]

[0443] TACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGA Attorney Docket No: 046483 -7482WO 1(04099)

[0444] CATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTC AAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTT GGCTGCATACGCGTCGTGAGGCTCCGGTGCCCGTCAGTGGGC AGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAG GGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGG GTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTT CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGC CGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACA GGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTAC GGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCT GCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGG GTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCC TCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCG CGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTT CGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGC GACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCA AGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGG CGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGC GGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAG TCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCG CCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCG GCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCC TGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAG AGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTT CCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGG GCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTTTTGGAGT ACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAG TTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCT TGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGT TGTTGTGTTATTTCTTCTTGTGCTCTACTATTTTCCATGCGATAGGTCCCGTTCGAAGGACCTAAGGTCGTGCTTTACGAAAAATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCT GCTCCACGCCGCCAGGCCGGGATCCGCGGCCCAGGCGGCCC AGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCT GGGGGGTCTCTAAAACTCTCCTGTGCAGCCTCTGGAAGCATC CACAGTATCAATGCCATGGGCTGGTACCGCCAGACTCCAGG GAACCAGCGCGAGTTGGTCGCAACTATTACTAATGGTGTTAA TGGTGTCACCACAAACTATGCAAACTCCGTGAAGGGCCGATT CACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGC AAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTAC TGTACACCAGACCCCCTGCGGTACCTGTTTGATTCCTGGGGC CAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCCACTAGT TCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCT GATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCT TGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGG TGAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAA CAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGA TGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGAT GTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCC GCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAA

[0445]

[0446] TCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGAC Attorney Docket No: 046483 -7482WO 1(04099)

[0447] GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAA GAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATA AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAG CGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCT CAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGCTAAGTCGACAATCAACCTCTGGATT ACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTG CTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTA TCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGT ATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCG TTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACG CAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCC TTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGA ACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCG GCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCT GACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATT CTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAAT CCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGG CCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCT CCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACC TCTTTTATTATGAAACACAAGAATAGAAACTGTGAGCGAGAGGAGCCTAGGGCATAGGTGAGGCATTACATTTTACGACCCTA CCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTC TCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAAC TAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAG TGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTA ACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCT CTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATA ACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGT TTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCA CAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTG TGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTC TAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCC GCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGA GGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG GCTTTTTTGGAGGCCTAGCTAGGGACGTACCCAATTCGCCCT ATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTAC AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATC GCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCG AAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGC CTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAG CGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACAC TTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTC CTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAAT CGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGT GGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTG GAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGA ACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAG GGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGA

[0448]

[0449] TTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGC Attorney Docket No: 046483 -7482WO 1(04099)

[0450] TTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC CCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCG CTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTT ATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCC AGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG

[0451] pTRPE-VHHB30- GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGT 28BBz AAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACA CTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGA AAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCA GTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTAC TTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTT TGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG AACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGAC ACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACT ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATT AATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTC TGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAAT CTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA CGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAG ATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTG TCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAA CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTG ATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCC ACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAG CGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAG GCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCG CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAG GGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGG AACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATG GAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTT TTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCT GATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTC TCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACA GGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATT AATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACAC

[0452]

[0453] TTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGA Attorney Docket No: 046483 -7482WO 1(04099)

[0454] TAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCC AAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGA GCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTT GCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGA GAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTG GTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGA CATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGAT ATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTC TGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTA GGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTG CTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAAC TAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCT AGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAA ACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAG CGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACG CCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGG GTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGC GATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAA ATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAG AACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAG GCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAG ACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGC AACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACA CCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAA AAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGA CCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATT ATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAG CACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAA AAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGG AGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGA CGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGC AGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTG TTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAG AATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCC TGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTG CTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAAC AGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAA ATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAA TCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGA ATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAAC AAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGG AGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATA GTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAG ACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGG AATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCC ATTCGATTAGTGAACGGATCTCGACGGTATCGATTAGACTGT AGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGG AAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATAT AGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACA GCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAA

[0455]

[0456] AACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTA Attorney Docket No: 046483 -7482WO 1(04099)

[0457] CAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAA TTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAA TCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAG AGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAG TATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGG TACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGA CATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTC AAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTT GGCTGCATACGCGTCGTGAGGCTCCGGTGCCCGTCAGTGGGC AGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAG GGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGG GTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTT CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGC CGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACA GGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTAC GGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCT GCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGG GTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCC TCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCG CGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTT CGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGC GACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCA AGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGG CGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGC GGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAG TCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCG CCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCG GCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCC TGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAG AGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTT CCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGG GCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTTTTGGAGT ACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAG TTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCT TGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGT TGTTGTGTTATTTCTTCTTGTGCTCTACTATTTTCCATGCGATAGGTCCCGTTCGAAGGACCTAAGGTCGTGCTTTACGAAAAATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCT GCTCCACGCCGCCAGGCCGGGATCCGCGGCCCAGGCGGCCC AGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCT GGGGGGTCTCTAAAACTCTCCTGTGCAGCCTCTGGAAGCATC CACAGTATCAATGCCATGGGCTGGTACCGCCAGACTCCAGG GAACCAGCGCGAGTTGGTCGCAACTATTACTAATGGTGTTAA TGGTGTCACCACAAACTATGCAAACTCCGTGAAGGGCCGATT CACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGC AAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTAC TGTACACCAGACCCCCTGCGGTACCTGTTTGATTCCTGGGGC CAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCCACTAGT TCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCT GATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCT

[0458]

[0459] TGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGG Attorney Docket No: 046483 -7482WO 1(04099)

[0460] TGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATG AACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTAC CAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC GCTAGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAA ACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA TGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCC CGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCA ATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGA CGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGA GCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTC TCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGCTAAGTCGACAATCAACCTCTGGATT ACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTG CTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTA TCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGT ATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCG TTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACG CAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCC TTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGA ACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCG GCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCT GACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATT CTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAAT CCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGG CCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCT CCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACC TCTTTTATTATGAAACACAAGAATAGAAACTGTGAGCGAGAGGAGCCTAGGGCATAGGTGAGGCATTACATTTTACGACCCTA CCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTC TCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAAC TAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAG TGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTA ACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCT CTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATA ACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGT TTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCA CAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTG TGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTC TAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCC GCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGA GGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG GCTTTTTTGGAGGCCTAGCTAGGGACGTACCCAATTCGCCCT ATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTAC AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATC GCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCG AAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGC CTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAG

[0461]

[0462] CGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACAC Attorney Docket No: 046483 -7482WO 1(04099)

[0463] TTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTC CTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAAT CGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGT GGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTG GAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGA ACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAG GGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGA TTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGC TTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC CCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCG CTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTT ATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCC AGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG CEA ATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTA VHHB30.28BBz / MSL ACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCA NVHHl. BB / Split BiCCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTG CAR Lentiviral CCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGA construct DNA CGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAG TGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAG ATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTG TGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCC CGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCT CTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAA GAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTG ACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCG TCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAA ATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAA ACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAG TTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAA ATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGA AGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTG TGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTT TAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCAC CGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGG AGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAAT ATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACC AAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAG TGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAG GAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAG GCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAA TTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACT CACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGG CTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATT TGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT TGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGG AATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAA

[0464]

[0465] TTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAA Attorney Docket No: 046483 -7482WO 1(04099)

[0466] CCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATA AATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGC TGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGG TAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATA GAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACC TCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAA GAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATT AGTGAACGGATCGGCACTGCGTGCGCCAATTCTGCAGACAA ATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGAT TGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAG CAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGAT CCAGTTTGGTTAGTACCGGGCCCGCTCTAGCGTGAGGCTCCG GTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTA GAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGT ACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATA TAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGT TTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCG GGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAA TTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGC TTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTT AAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTG GGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGC GCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATT TTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCT TGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTT TGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCA CATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGA ATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTG CCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCA AGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATG GCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGA CGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAA AGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGAC TCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTC TCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGG TTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACT GAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAA TTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTC AGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTG AGCGGCCGCTGAGTTAACTATTCTAGAGCCAGCATGGCCTTA CCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCAC GCCGCTCGACCAGCGGCCCAGGCGGCCCAGGTACAACTGCA GGAGAGCGGCGGCGGTCTTGTCCAAGCAGGTGGCTCACTCA AACTCTCTTGTGCAGCCAGTGGATCAATACATTCTATTAACG CTATGGGGTGGTATAGGCAAACACCCGGCAATCAGCGGGAA CTGGTAGCCACAATTACAAACGGTGTAAATGGAGTTACCAC GAATTACGCTAATTCCGTCAAGGGCCGATTTACCATTTCACG GGACAATGCAAAGAATACCGTCTATTTGCAGATGAACAGCC

[0467]

[0468] TCAAGCCGGAAGACACCGCGGTTTATTATTGCACCCCCGATC Attorney Docket No: 046483 -7482WO 1(04099)

[0469] CGCTTAGGTACCTTTTTGATTCTTGGGGCCAGGGTACCCAGG TTACTGTATCATCCGGCCAGGCCGGCCAGGAGTCTAAATACG GCCCGCCTTGTCCGCCATGTCCATTTTGGGTGTTGGTGGTCGT AGGCGGAGTCCTTGCGTGTTACTCCCTGTTGGTAACCGTTGC TTTTATAATTTTTTGGGTGCGGTCTAAGCGAAGTCGCTTGCTC CACAGTGATTATATGAACATGACGCCGCGCAGACCCGGGCC TACCAGAAAGCATTACCAGCCGTATGCGCCGCCTAGGGACTT TGCTGCATATAGATCCAGAGTGAAGTTTTCCCGATCAGCAGA CGCCCCAGCGTATCAACAGGGACAAAATCAGTTGTACAACG AGCTGAATCTCGGTCGCAGAGAAGAGTATGATGTGCTCGAC AAAAGACGAGGGCGGGACCCTGAAATGGGCGGGAAACCTA GAAGGAAAAACCCACAAGAAGGGCTGTACAATGAGCTTCAA AAAGATAAAATGGCAGAGGCATACAGTGAAATAGGCATGAA AGGGGAGCGCCGGCGAGGAAAAGGCCACGACGGACTGTATC AAGGTCTTAGTACTGCTACGAAGGACACCTACGACGCGTTGC ATATGCAAGCATTGCCCCCCAGAATAGACAGGAGAAAACGC GGAAGTGGGGCAACAAACTTCTCTCTTCTTAAGCAAGCGGG AGACGTGGAAGAGAACCCGGGACCAGGATCTGGGATGGCTT TGCCCGTTACTGCACTTCTCCTTCCACTCGCACTTCTCTTGCA CGCAGCCCGGCCGGGATCCCAGGTGCAGCTGCAGGAGTCTG GAGGAGGCTCGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCT GTGCAGCCTCTGGATTCACCTTCAGTATGTCTGCCATGAACT GGGTCCGCCAAACTGCAGGCAAGGGGCTCGAGTGGGTCTCC GGTATTACTAATAGTGGTGGATTTACAAGCTATGCAGACTCC GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CACGCTGTATCTGCAAATGAATAGCCTGAAACCTGAGGACA CGGCCGTGTATTACTGTACAAGATTTGCTAGTTCGTTATCTCC ACCCGTGAGCCAGGGGACCCAGGTCACCGTCTCCTCATCCGG AGAATCAAAATATGGTCCCCCATGTCCACCTTGTCCATTTTG GGTTCTTGTTGTAGTTGGAGGCGTCTTGGCGTGTTATAGCTT GCTCGTCACTGTTGCATTTATAATATTCTGGGTAAAGCGGGG TAGGAAAAAACTGCTCTATATCTTCAAGCAGCCTTTTATGAG GCCGGTTCAAACCACCCAAGAGGAGGATGGATGTAGCTGTC GATTTCCAGAAGAGGAAGAGGGCGGTTGCGAACTCTGAGTC GACAACCAACCACTTGATTACAAAATTTGTGAAAGATTGACT GGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACG CTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGC TTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTT ATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGT GCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTG CCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCT CCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCG CTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGT GGTGTTGTCGGGGAAGCTGACGTCCTTTCCTTGGCTGCTCGC CTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTA CGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGG CCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGC CCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCT GGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGG CAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGA

[0470]

[0471] CTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTT Attorney Docket No: 046483 -7482WO 1(04099)

[0472] TTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCT GGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTC AATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTC TGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTT AGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATC TTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAG AGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACA AATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTT TTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGT ATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGC CCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTT TATTTATGCAGAGGCCGAGGACGCCTCGGCCTCTGAGCTATT CCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGCTAGGG ACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGTTTCCA TAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTC AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACC AGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCC GACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCG GGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTC AGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAAC TATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCA CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTA TGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA CGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCT GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC CGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTG CAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAG ATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACG AAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTA AATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTT ACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTC TATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGAT AACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGC AATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATC AGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGT GGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCT CGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGAT CAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCG GTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTG GCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAAT TCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTG GTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC GACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCG CGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAA CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTG AGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCT

[0473]

[0474] TCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAA Attorney Docket No: 046483 -7482WO 1(04099)

[0475] ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGA CACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTA TTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT ATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGC GCACATTTCCCCGAAAAGTGCCACCTGACGCGGATCGGGAG ATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCT GATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTG TTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTAC AACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGC TTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCA GATATACGCGCAATTAACCCTCACTAAAGGGAACAAAAGCT GGAGCTGCAAGCTTA CEA ATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTA VHHB30.28BBz / MSL ACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCA N VHH12. BB / Split BiCCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTG CAR Lentiviral CCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGA construct DNA CGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAG TGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAG ATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTG TGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCC CGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCT CTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAA GAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTG ACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCG TCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAA ATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAA ACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAG TTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAA ATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGA AGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTG TGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTT TAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCAC CGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGG AGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAAT ATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACC AAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAG TGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAG GAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAG GCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAA TTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACT CACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGG CTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATT TGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT TGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGG AATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAA TTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAA CCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATA AATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGC

[0476]

[0477] TGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGG Attorney Docket No: 046483 -7482WO 1(04099)

[0478] TAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATA GAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACC TCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAA GAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATT AGTGAACGGATCGGCACTGCGTGCGCCAATTCTGCAGACAA ATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGAT TGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAG CAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGAT CCAGTTTGGTTAGTACCGGGCCCGCTCTAGCGTGAGGCTCCG GTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTA GAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGT ACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATA TAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGT TTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCG GGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAA TTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGC TTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTT AAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTG GGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGC GCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATT TTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCT TGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTT TGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCA CATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGA ATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTG CCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCA AGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATG GCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGA CGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAA AGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGAC TCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTC TCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGG TTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACT GAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAA TTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTC AGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTG AGCGGCCGCTGAGTTAACTATTCTAGAGCCAGCATGGCCTTA CCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCAC GCCGCTCGACCAGCGGCCCAGGCGGCCCAGGTACAACTGCA GGAGAGCGGCGGCGGTCTTGTCCAAGCAGGTGGCTCACTCA AACTCTCTTGTGCAGCCAGTGGATCAATACATTCTATTAACG CTATGGGGTGGTATAGGCAAACACCCGGCAATCAGCGGGAA CTGGTAGCCACAATTACAAACGGTGTAAATGGAGTTACCAC GAATTACGCTAATTCCGTCAAGGGCCGATTTACCATTTCACG GGACAATGCAAAGAATACCGTCTATTTGCAGATGAACAGCC TCAAGCCGGAAGACACCGCGGTTTATTATTGCACCCCCGATC CGCTTAGGTACCTTTTTGATTCTTGGGGCCAGGGTACCCAGG TTACTGTATCATCCGGCCAGGCCGGCCAGGAGTCTAAATACG

[0479]

[0480] GCCCGCCTTGTCCGCCATGTCCATTTTGGGTGTTGGTGGTCGT Attorney Docket No: 046483 -7482WO 1(04099)

[0481] AGGCGGAGTCCTTGCGTGTTACTCCCTGTTGGTAACCGTTGC TTTTATAATTTTTTGGGTGCGGTCTAAGCGAAGTCGCTTGCTC CACAGTGATTATATGAACATGACGCCGCGCAGACCCGGGCC TACCAGAAAGCATTACCAGCCGTATGCGCCGCCTAGGGACTT TGCTGCATATAGATCCAGAGTGAAGTTTTCCCGATCAGCAGA CGCCCCAGCGTATCAACAGGGACAAAATCAGTTGTACAACG AGCTGAATCTCGGTCGCAGAGAAGAGTATGATGTGCTCGAC AAAAGACGAGGGCGGGACCCTGAAATGGGCGGGAAACCTA GAAGGAAAAACCCACAAGAAGGGCTGTACAATGAGCTTCAA AAAGATAAAATGGCAGAGGCATACAGTGAAATAGGCATGAA AGGGGAGCGCCGGCGAGGAAAAGGCCACGACGGACTGTATC AAGGTCTTAGTACTGCTACGAAGGACACCTACGACGCGTTGC ATATGCAAGCATTGCCCCCCAGAATAGACAGGAGAAAACGC GGAAGTGGGGCAACAAACTTCTCTCTTCTTAAGCAAGCGGG AGACGTGGAAGAGAACCCGGGACCAGGATCTGGGATGGCTT TGCCCGTTACTGCACTTCTCCTTCCACTCGCACTTCTCTTGCA CGCAGCCCGGCCGGGATCCCAGGTGCAGCTGCAGGAGTCTG GAGGAGGATTGGTGCAGACTGGGGGCTCTCTGAGACTCTCCT GTGCAGCCTCTAGAAGTATTTTCAGTACATATGCCATGGGCT GGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCG GCTATTGGTTATAGTCATGGAAACACAGACTATGTAGACTCC GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CACGATGTATCTGCAAATGAACAGCCTGAGACCTGAGGACA CGGCCGTTTATTACTGTGCAGCATACACAGGACCTCGGCCAC CACGGCAGGCGCCCTTATATGACTACTGGGGCCAGGGGACC CAGGTCACCGTCTCCTCATCCGGAGAATCAAAATATGGTCCC CCATGTCCACCTTGTCCATTTTGGGTTCTTGTTGTAGTTGGAG GCGTCTTGGCGTGTTATAGCTTGCTCGTCACTGTTGCATTTAT AATATTCTGGGTAAAGCGGGGTAGGAAAAAACTGCTCTATA TCTTCAAGCAGCCTTTTATGAGGCCGGTTCAAACCACCCAAG AGGAGGATGGATGTAGCTGTCGATTTCCAGAAGAGGAAGAG GGCGGTTGCGAACTCTGAGTCGACAACCAACCACTTGATTAC AAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCT CCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATC ATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTA TAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTT GTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCA ACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTT TCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAA CTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGG CTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTG ACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTC TGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATC CAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGC CTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTC CCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACCT TTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCAC TTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTC CCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCT CTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACT

[0482]

[0483] AGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT Attorney Docket No: 046483 -7482WO 1(04099)

[0484] GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAA CTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTC TAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAA CTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTT TATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCAC AAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT GGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCT AGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCC GCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGA GGACGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG GCTTTTTTGGAGGCCTAGCTAGGGACGTACCCAATTCGCCCT ATAGTGAGTCGTATTACGTTTCCATAGGCTCCGCCCCCCTGA CGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAA ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCG TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGC CCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACT GGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTAC AGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAA GAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT TCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATT ACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTA AGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTA GATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAG TATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAAT CAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATC CATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACG GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCA GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTT TATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA GAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTG CCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTA CATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCG GTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTAT CACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT CTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC AGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGG CGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCG ATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTT ACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCA AAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT

[0485]

[0486] GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTA Attorney Docket No: 046483 -7482WO 1(04099)

[0487] TCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCC GAAAAGTGCCACCTGACGCGGATCGGGAGATCTCCCGATCC CCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATA GTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGC TGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAG GCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGC GTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGCA ATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGC TTA CEACAM5- ATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTA VHHB30 / MSLN- ACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCA VHH1.28BBz / Tandcm CCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTG Bi-CAR Lentiviral CCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGA construct DNA CGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAG TGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAG ATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTG TGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCC CGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCT CTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAA GAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTG ACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCG TCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAA ATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAA ACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAG TTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAA ATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGA AGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTG TGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTT TAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCAC CGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGG AGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAAT ATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACC AAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAG TGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAG GAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAG GCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAA TTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACT CACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGG CTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATT TGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT TGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGG AATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAA TTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAA CCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATA AATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGC TGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGG TAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATA

[0488]

[0489] GAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACC Attorney Docket No: 046483 -7482WO 1(04099)

[0490] TCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAA GAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATT AGTGAACGGATCGGCACTGCGTGCGCCAATTCTGCAGACAA ATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGAT TGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAG CAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGAT CCAGTTTGGTTAGTACCGGGCCCGCTCTAGCGTGAGGCTCCG GTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTA GAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGT ACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATA TAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGT TTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCG GGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAA TTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGC TTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTT AAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTG GGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGC GCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATT TTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCT TGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTT TGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCA CATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGA ATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTG CCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCA AGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATG GCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGA CGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAA AGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGAC TCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTC TCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGG TTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACT GAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAA TTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTC AGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTG AGCGGCCGCTGAGTTAACTATTCTAGAGCCAGCATGGCCTTA CCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCAC GCCGCCAGGCCGGCGGCCCAGGCGGCCGGATCCCAGGTGCA ATTGCAAGAGAGCGGAGGCGGGCTTGTCCAGGCCGGTGGGT CTCTCAAACTTTCTTGCGCCGCGTCTGGTAGTATACATTCAAT CAACGCAATGGGATGGTATCGGCAAACACCCGGCAACCAAC GGGAGCTCGTAGCGACCATTACAAATGGTGTCAATGGCGTG ACCACCAACTACGCGAACTCTGTCAAAGGACGCTTTACTATA TCTAGGGATAACGCGAAGAACACCGTTTACCTGCAGATGAA TAGTCTTAAGCCTGAAGATACAGCGGTATATTACTGCACACC TGATCCGCTGCGCTACTTGTTTGACAGCTGGGGCCAAGGGAC GCAAGTCACAGTCAGCTCACATATGGGCTCCGGCAGTGGGT CAGGATCAGGTTCAGGTAGCGGGTCAGGTTCCGCTAGCCAG GTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGCCTGG

[0491]

[0492] GGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTT Attorney Docket No: 046483 -7482WO 1(04099)

[0493] CAGTATGTCTGCCATGAACTGGGTCCGCCAAACTGCAGGCA AGGGGCTCGAGTGGGTCTCCGGTATTACTAATAGTGGTGGAT TTACAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCT CCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAAT AGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTACAAG ATTTGCTAGTTCGTTATCTCCACCCGTGAGCCAGGGGACCCA GGTCACCGTCTCCTCATCCGGAGGCCAGGCCGGCCAGGAGA GCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTTTTTGGGTGCT GGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGT AACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAG CAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCG CCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACC ACGCGACTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGA AACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTAC AAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCA GAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCA GCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTA CGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG GGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTG AGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCA CGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACA CCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAG TCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGA CTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATA CGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATG GCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTC TTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGG TGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCA TTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCC CCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGC CCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTC CGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCTTGGCTGCT CGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTG CTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCG CGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTT CGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCG CCTGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACA AGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGG GGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCT GCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGA GCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAG CCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCC CGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC TTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGT CATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATA TCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGT TACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATC

[0494]

[0495] AATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAAC Attorney Docket No: 046483 -7482WO 1(04099)

[0496] TCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAAT TTTTTTTATTTATGCAGAGGCCGAGGACGCCTCGGCCTCTGA GCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGC TAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGT TTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTC CCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGG TATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTA TCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGC GAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGC CTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCG CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT CTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTT TTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTA TCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCT GACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCG ATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCG TGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCA GTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCG CAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTAT TAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAA TAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGT GTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAA AAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT AAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTG TGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTA TGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATA ATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTG GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGC TGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT GATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGC AAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGA TACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTT CCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGGATCG GGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCT GCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTT GTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAA GCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAA

[0497]

[0498] TCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACG Attorney Docket No: 046483 -7482WO 1(04099)

[0499] GGCCAGATATACGCGCAATTAACCCTCACTAAAGGGAACAA AAGCTGGAGCTGCAAGCTTA CEACAM5- ATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTA VHHB30 / MSLN- ACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCA VHH12.28BBz / Tande CCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTG m Bi-CAR Lentiviral CCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGA construct DNA CGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAG TGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAG ATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTG TGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCC CGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCT CTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAA GAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTG ACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCG TCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAA ATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAA ACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAG TTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAA ATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGA AGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTG TGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTT TAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCAC CGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGG AGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAAT ATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACC AAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAG TGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAG GAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAG GCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAA TTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACT CACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGG CTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATT TGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT TGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGG AATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAA TTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAA CCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATA AATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGC TGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGG TAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATA GAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACC TCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAA GAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATT AGTGAACGGATCGGCACTGCGTGCGCCAATTCTGCAGACAA ATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGAT TGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAG CAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGAT

[0500]

[0501] CCAGTTTGGTTAGTACCGGGCCCGCTCTAGCGTGAGGCTCCG Attorney Docket No: 046483 -7482WO 1(04099)

[0502] GTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTA GAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGT ACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATA TAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGT TTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCG GGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAA TTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGC TTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTT AAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTG GGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGC GCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATT TTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCT TGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTT TGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCA CATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGA ATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTG CCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCA AGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATG GCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGA CGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAA AGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGAC TCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTC TCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGG TTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACT GAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAA TTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTC AGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTG AGCGGCCGCTGAGTTAACTATTCTAGAGCCAGCATGGCCTTA CCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCAC GCCGCCAGGCCGGCGGCCCAGGCGGCCGGATCCCAGGTGCA ATTGCAAGAGAGCGGAGGCGGGCTTGTCCAGGCCGGTGGGT CTCTCAAACTTTCTTGCGCCGCGTCTGGTAGTATACATTCAAT CAACGCAATGGGATGGTATCGGCAAACACCCGGCAACCAAC GGGAGCTCGTAGCGACCATTACAAATGGTGTCAATGGCGTG ACCACCAACTACGCGAACTCTGTCAAAGGACGCTTTACTATA TCTAGGGATAACGCGAAGAACACCGTTTACCTGCAGATGAA TAGTCTTAAGCCTGAAGATACAGCGGTATATTACTGCACACC TGATCCGCTGCGCTACTTGTTTGACAGCTGGGGCCAAGGGAC GCAAGTCACAGTCAGCTCACATATGGGCTCCGGCAGTGGGT CAGGATCAGGTTCAGGTAGCGGGTCAGGTTCCGCTAGCCAG GTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGACTGG GGGCTCTCTGAGACTCTCCTGTGCAGCCTCTAGAAGTATTTT CAGTACATATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGA AGGAGCGTGAGTTTGTAGCGGCTATTGGTTATAGTCATGGAA ACACAGACTATGTAGACTCCGTGAAGGGCCGATTCACCATCT CCAGAGACAACGCCAAGAACACGATGTATCTGCAAATGAAC AGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCA TACACAGGACCTCGGCCACCACGGCAGGCGCCCTTATATGA CTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATCCGG

[0503]

[0504] AGGCCAGGCCGGCCAGGAGAGCAAGTACGGCCCTCCCTGCC Attorney Docket No: 046483 -7482WO 1(04099)

[0505] CCCCTTGCCCTTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCT GGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTC TGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTA CATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGC ATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATC GCTCCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAA CAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGA TGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGAT GTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCC GCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAA TCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGAC GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAA GAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATA AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAG CGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCT CAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGCTAAGTCGACAATCAACCTCTGGATT ACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTG CTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTA TCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGT ATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCG TTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACG CAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCC TTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGA ACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCG GCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCT GACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATT CTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAAT CCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGG CCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCT CCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACC TCTTTTATTATGAAACACAAGAATAGAAACTGTGAGCGAGAGGAGCCTAGGGCATAGGTGAGGCATTACATTTTACGACCCTA CCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTC TCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAAC TAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAG TGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTA ACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCT CTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATA ACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGT TTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCA CAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTG TGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTC TAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCC GCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGA GGACGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG GCTTTTTTGGAGGCCTAGCTAGGGACGTACCCAATTCGCCCT ATAGTGAGTCGTATTACGTTTCCATAGGCTCCGCCCCCCTGA CGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAA ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA

[0506]

[0507] AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG Attorney Docket No: 046483 -7482WO 1(04099)

[0508] GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCG TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGC CCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACT GGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTAC AGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAA GAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT TCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATT ACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTA AGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTA GATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAG TATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAAT CAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATC CATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACG GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCA GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTT TATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA GAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTG CCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTA CATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCG GTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTAT CACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT CTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC AGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGG CGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCG ATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTT ACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCA AAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTA TCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCC GAAAAGTGCCACCTGACGCGGATCGGGAGATCTCCCGATCC CCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATA GTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGC TGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAG GCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGC GTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGCA ATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGC TTA EF 1 alpha promoter CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCG CCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTG AACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAA GTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGG

[0509]

[0510] GAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTT Attorney Docket No: 046483 -7482WO 1(04099)

[0511] TTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGT GTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTT GCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTC TTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCG AGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTT GAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTG GTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTA GCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCT GGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACT GGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGT GCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGC GCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCC GGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCC CGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCG TGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAG CTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTG AGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCC GTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGG CACCTCGATTAGTTCTCGTGCTTTTGGAGTACGTCGTCTTTAG GTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTG AGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATG TAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGT TCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCC ATTTCAGGTGTCGTGA RSV promoter ATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTA ACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCA CCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTG CCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGA CGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAG TGCCTAGCTCGATACATAAAC

[0512] 5’ SIN LTRDNA GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCT GGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTG CCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGAC TCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGG AAAATCTCTAGCAG HIV partial gag CGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCT sequence (psi / RRE) CTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAA DNA GAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTG ACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCG TCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAA ATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAA ACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAG TTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAA ATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGA AGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTG TGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTT TAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCAC CGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGA GATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAA AGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGG

[0513]

[0514] CAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGG Attorney Docket No: 046483 -7482WO 1(04099)

[0515] AATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAG CACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCA GACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGC TGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACA GTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGT GGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGG GTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGA ATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATC ACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTAC ACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCA GCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAAT GGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGT GGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAG GTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAG TTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCC CAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGA AGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAG TGAACGGATCTCGACGGTAT HIV central polypurine TAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAG tract (cPPT) DNA GAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATA TAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAAC AGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAA AAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACT ACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGA ATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGA ATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAA GAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCA GTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGG GTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAG ACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATT CAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTT TGGCT

[0516] Woodchuck Hepatitis ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTA Virus Post- TTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGC transcriptional TTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTC Regulatory Element ATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATG (WPRE) AGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGC ACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCC ACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCC CTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTG GTGTTGTCGGGGAAGCTGACGTCCTTTCCTTGGCTGCTCGCC TGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTAC GTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGC CTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCC CTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG

[0517] 3’ SIN LTR(U3 del) TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTT TTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCT GGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTC AATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTC

[0518]

[0519] TGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTT Attorney Docket No: 046483 -7482WO 1(04099)

[0520] AGTCAGTGTGGAAAATCTCTAGCAG

[0521] Hinge / Linker (GSGGS)n, where n represents an integer of at least 1 Hinge / Linker (GGGS)n, where n represents an integer of at least 1 Hinge / Linker (GGGGS)n, where n represents an integer of at least 1 Hinge / Linker GGSG

[0522] Hinge / Linker GGSGG

[0523] Hinge / Linker GSGSG

[0524] Hinge / Linker GSGGG

[0525] Hinge / Linker GGGSG

[0526] Hinge / Linker GSSSG

[0527] Hinge / Linker GGGGS

[0528] Hinge / Linker (G4S)3

[0529] Hinge / Linker (G4S)4

[0530] Hinge DKTHT

[0531] Hinge CPPC

[0532] Hinge CPEPKSCDTPPPCPR

[0533] Hinge ELKTPLGDTTHT

[0534] Hinge KSCDKTHTCP

[0535] Hinge KCCVDCP

[0536] Hinge KYGPPCP

[0537] Hinge EPKSCDKTHTCPPCP

[0538] Hinge ERKCCVECPPCP

[0539] Hinge ELKTPLGDTTHTCPRCP

[0540] Hinge SPNMVPHAHHAQ

[0541] T2A Peptide EGRGSLLTCGDVEENPGP

[0542] P2A Peptide ATNFSLLKQAGDVEENPGP

[0543] E2A Peptide GSGQCTNYALLKLAGDVESNPGP

[0544] F2A Peptide GSGVKQTLNFDLLKLAGDVESNPGP

[0545] EMCV IRES GGGCGGCTATAGGGGCGGCTCGAGCGGGATCAATTCCGCCC CCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCG GTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCGCCCG GAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCT TTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGT GAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAA CGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTG GCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGAT ACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAG TTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGT ATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATT GTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACAT GTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACC ACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATG GCCACAACC

[0546] BamHl-Forward primer CGCATTGGATCCCAGGTGCAGCTGCAGGAGTCT

[0547] EF-la primer TCAAGCCTCAGACAGTGGTTC

[0548] WPRE-Reverse primer GCGTATCCACATAGCGTAAA

[0549] Sfi [-Forward primer GGCGGCCCAGGCGGCCCAGGTG

[0550] Sfil-Reverse primer TGAGGAGACGGTGACCTGCCTGGCCGGCCTGGCC

[0551]

[0552] BspEI-Rcvcrsc primer ATTTCCGGATGAGGAGACGGTGACCT Attorney Docket No: 046483 -7482WO 1(04099)

[0553] 171 BmtI / Nhel-Forward GGGGCTAGCCAGGTGCAGCTGCAGGAGT

[0554] primer

[0555] 172 Ndel-Reverse primer GCCCATATGTGAGGAGACGGTGACCTG

[0556] 173 pComb3x- Forward GCTCACTCATTAGGCACCCCA

[0557] primer

[0558] 174 pComb3x- Reverse CACCACCCTCCTAAGAAGCG

[0559] primer

[0560] 175 CALL001 primer GTCCTGGCTGCTCTTCTACAAGG

[0561] 176 CALL002 primer GGTACGTGCTGTTGAACTGTTGG

[0562] 177 FR1-RSCF primer GTCCTGGCTGCTCTTCTACAAGG

[0563]

[0564] 178 VHH-RSCB primer GGTACGTGCTGTTGAACTGTTGG

[0565] Methods of Generating Modified Immune Cells

[0566] The present invention provides methods for producing / generating a modified immune cell or precursor cell thereof (e.g., a T cell, NK, NKT, gamma-delta T cell). The cells are engineered by introducing a nucleic acid encoding the MSLN CAR, CEACAM5 CAR, MSLN / CEACAM5 bi-CAR and / or additional proteins of interest. In some embodiments, the CAR may be accompanied by a gene modification system that is capable of modifying the expression or sequence of an endogenous gene.

[0567] Methods of introducing nucleic acids or expression constructs into a cell include physical, biological, and chemical methods. Physical methods for introducing a polynucleotide, such as RNA, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods which include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, MA) or the Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendorf, Hamburg Germany). RNA can also be introduced into cells using cationic liposome mediated transfection using lipofection, using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

[0568] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpesviruses, adenoviruses, and adeno-associated viruses, and the like. See, for example, U. S. Pat. Nos. 5,350,674 and 5,585,362. Attorney Docket No: 046483 -7482WO 1(04099)

[0569] In some embodiments, a nucleic acid encoding the MSLN CAR, CEACAM5 CAR, or MSLN / CEACAM5 bi-CAR of the invention is introduced into a cell by an expression vector. Expression vectors comprising a nucleic acid encoding the CAR are provided herein. Suitable expression vectors include lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon mediated vectors, such as Sleeping Beauty, Piggyback, and Integrases such as Phi31. Other suitable expression vectors include Epstein-Barr virus (EBV), herpes simplex virus (HSV), and retrovirus expression vectors.

[0570] Adenovirus expression vectors are based on adenoviruses, which have a low capacity for integration into genomic DNA but a high efficiency for transfecting host cells. Adenovirus expression vectors contain adenovirus sequences sufficient to: (a) support packaging of the expression vector and (b) to ultimately express the MSLN CAR, CEACAM5 CAR, or MSLN / CEACAM5 bi-CAR in the host cell. In some embodiments, the adenovirus genome is a 36 kb, linear, double stranded DNA, where a foreign DNA sequence e.g., a nucleic acid encoding the CAR) may be inserted to substitute large pieces of adenoviral DNA in order to make the expression vector of the present invention (see, e.g., Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).

[0571] Another expression vector is based on an adeno associated virus, which takes advantage of the adenovirus coupled systems. This AAV expression vector has a high frequency of integration into the host genome. It can infect non-dividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue cultures or in vivo. The AAV vector has a broad host range for infectivity. Details concerning the generation and use of AAV vectors are described in U. S. Patent Nos. 5,139,941 and 4,797,368.

[0572] Retrovirus expression vectors are capable of integrating into the host genome, delivering a large amount of foreign genetic material, infecting a broad spectrum of species and cell types, and being packaged in special cell lines. The retrovirus vector is constructed by inserting a nucleic acid (e.g., a nucleic acid encoding the CAR) into the viral genome at certain locations to produce a virus that is replication defective. Though the retrovirus vectors are able to infect a broad variety of cell types, integration, and stable expression of the CAR, requires the division of host cells. Attorney Docket No: 046483 -7482WO 1(04099)

[0573] Lentivirus vectors are derived from lentiviruses, which are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (see, e.g., U. S. Patent Nos. 6,013,516 and 5,994, 136). Some examples of lentiviruses include the human immunodeficiency viruses (HIV-1, HIV-2) and the simian immunodeficiency virus (SIV). Lentivirus vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentivirus vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, e.g., of a nucleic acid encoding the MSLN CAR, CEACAM5 CAR, or MSLN / CEACAM5 bi-CAR (see, e.g., U. S. Patent No. 5,994,136).

[0574] Expression vectors including a nucleic acid of the present disclosure can be introduced into a host cell by any means known to persons skilled in the art. The expression vectors may include viral sequences for transfection, if desired. Alternatively, the expression vectors may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. The host cell may be grown and expanded in culture before introduction of the expression vectors, followed by the appropriate treatment for introduction and integration of the vectors. The host cells are then expanded and may be screened by virtue of a marker present in the vectors.

[0575] Various markers that may be used are known in the art, and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. In some embodiments, the host cell is an immune cell or precursor thereof, e.g., a T cell, an NK cell, or an NKT cell.

[0576] The present invention also provides genetically engineered cells which include and stably express the MSLN CAR, CEACAM5 CAR, or MSLN / CEACAM5 bi-CAR of the present disclosure. In some embodiments, the genetically engineered cells are genetically engineered T-lymphocytes (T cells), regulatory T cells (Tregs), naive T cells (TN), memory T cells (for example, central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), natural killer T cells (NKT cells) and macrophages capable of giving rise to therapeutically relevant progeny. In one embodiment, the genetically engineered cells are autologous cells.

[0577] Modified cells (e.g., comprising the CAR) may be produced by stably transfecting host cells with an expression vector including a nucleic acid of the present disclosure. Additional Attorney Docket No: 046483 -7482WO 1(04099)

[0578] methods to generate a modified cell of the present disclosure include, without limitation, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and / or hydrodynamic delivery) and / or particle-based methods e.g., impalefection, using a gene gun and / or magnetofection). Transfected cells expressing the CAR of the present disclosure may be expanded ex vivo.

[0579] Physical methods for introducing an expression vector into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells including vectors and / or exogenous nucleic acids are well-known in the art. See, e.g., Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0580] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0581] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology' 5: 505-10).

[0582] However, compositions that have different structures in solution than the normal vesicular Attorney Docket No: 046483 -7482WO 1(04099)

[0583] structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0584] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the nucleic acids in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0585] Moreover, the nucleic acids may be introduced by any means, such as transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells. One nucleic acid may be introduced by one method and another nucleic acid may be introduced into the T cell by a different method.

[0586] Sources of Immune Cells

[0587] Prior to expansion, a source of immune cells is obtained from a subject for ex vivo manipulation. Sources of target cells for ex vivo manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow. For example, the source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject’s bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In certain exemplary embodiments, the subject is a human.

[0588] Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells and / or NKT cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In certain aspects, the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic Attorney Docket No: 046483 -7482WO 1(04099)

[0589] and / or autologous. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen.

[0590] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a gamma-delta T cell, a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), a natural killer T cell (NK cell) or a dendritic cell. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In an embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell or a hematopoietic stem cell.

[0591] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and gamma / delta T cells. In certain embodiments, any number of T cell lines available in the art, may be used.

[0592] In some embodiments, the methods include isolating immune cells from the subject, preparing, processing, culturing, and / or engineering them. In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for Attorney Docket No: 046483 -7482WO 1(04099)

[0593] engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g, transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0594] In certain aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0595] In some embodiments, the transduced cells are gamma-delta T cells.

[0596] Wang et al., J Imnnmother Cancer. 2021; 9(12): e003339 and U. S. Patent Publication No. 2024 / 197876 Al are incorporated herein by reference for their disclosures of compositions and methods for isolating gamma-delta T cells from a subject, and preparing, processing, culturing, and / or engineering them.

[0597] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, and pig. In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove Attorney Docket No: 046483 -7482WO 1(04099)

[0598] unwanted components, enrich for desired components, and lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0599] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in certain aspects, contain lymphocytes, including T.cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in certain aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In certain embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of a blood cell sample are removed, and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0600] In one embodiment, immune cells are obtained from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or another saline solution with or without buffer. In some embodiments, the undesirable Attorney Docket No: 046483 -7482WO 1(04099)

[0601] components of the apheresis sample may be removed, and the cells directly resuspended in culture media.

[0602] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in certain aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In certain aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0603] In certain exemplary embodiments, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In certain exemplary embodiments, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can Attorney Docket No: 046483 -7482WO 1(04099)

[0604] simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0605] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (marker111811) of one or more particular markers, such as surface markers, or that are negative for (marker) or express relatively low levels (marker1"") of one or more markers. For example, in certain aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD 127, and / or CD62L and / or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In certain exemplary embodiments, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g, DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0606] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In certain aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long- Attorney Docket No: 046483 -7482WO 1(04099)

[0607] term survival, expansion, and / or engraftment following administration, which in certain aspects is particularly robust in such sub-populations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0608] In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cell population and / or a CD8+ T population is enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in certain aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In certain aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in certain aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0609] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a Attorney Docket No: 046483 -7482WO 1(04099)

[0610] magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection.

[0611] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In certain aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agents include IL -2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0612] In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from an umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.

[0613] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD 14, CD 19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody. Attorney Docket No: 046483 -7482WO 1(04099)

[0614] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. An exemplary method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD1 lb, CD 16, HLA-DR, and CD8.

[0615] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g, particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0616] T cells can also be frozen after the washing step, which does not require the monocyteremoval step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[0617] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In Attorney Docket No: 046483 -7482WO 1(04099)

[0618] another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0619] Expansion of Immune Cells

[0620] Whether prior to or after modification of cells to express the MSLN CAR, CEACAM5 CAR, or MSLN / CEACAM5 bi-CAR, the cells can be activated and expanded in number using methods as described, for example, in U. S. Patent Nos. 6,352,694; 6,534,055; 6,905,680;

[0621] 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566;

[0622] 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U. S. Publication No. 20060121005. For example, the immune cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co- stimulatory molecule on the surface of the immune cells. In particular, immune cell populations may be stimulated by contact with an anti-CD3 antibody, or an antigenbinding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of an accessory molecule on the surface of the immune cells, a ligand that binds the accessory molecule is used. For example, immune cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the immune cells. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

[0623] Expanding the immune cells by the methods disclosed herein can be multiplied by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700 fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the immune cells expand in the range of about 20-fold to about 50-fold. Attorney Docket No: 046483 -7482WO 1(04099)

[0624] Following culturing, the immune cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. In certain exemplary embodiments, the level of confluence is 70% or greater before passing the cells to another culture apparatus. In particularly exemplary embodiments, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The immune cell medium may be replaced during the culture of the immune cells at any time. In certain exemplary embodiments, the immune cell medium is replaced about every 2 to 3 days. The immune cells are then harvested from the culture apparatus whereupon the immune cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cry opreserving the expanded immune cells. The cryopreserved immune cells are thawed prior to introducing nucleic acids into the immune cell.

[0625] In another embodiment, the method comprises isolating immune cells and expanding the immune cells. In another embodiment, the invention further comprises cry opreserving the immune cells prior to expansion. In yet another embodiment, the cryopreserved immune cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.

[0626] Another procedure for ex vivo expansion cells is described in U. S. Pat. No. 5,199,942 (incorporated herein by reference). Expansion, such as described in U. S. Pat. No. 5,199,942 can be an alternative or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of immune cells comprises the addition to the cellular growth factors, such as those described in U. S. Pat. No. 5,199,942, or other factors, such as Flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, expanding the immune cells comprises culturing the immune cells with a factor selected from the group consisting of Flt3-L, IL-1, IL-3, and c-kit ligand.

[0627] The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0628] Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition. A primary cell culture is a Attorney Docket No: 046483 -7482WO 1(04099)

[0629] culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.

[0630] Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (Pl or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging. Therefore, the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.

[0631] In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for immune cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of immune cells.

[0632] Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under Attorney Docket No: 046483 -7482WO 1(04099)

[0633] conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere e.g., air plus 5% CO2).

[0634] The medium used to culture the immune cells may include an agent that can co-stimulate the immune cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. This is because, as demonstrated by the data disclosed herein, a cell isolated by the methods disclosed herein can be expanded approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater. In one embodiment, the immune cells expand in the range of about 2-fold to about 50-fold, or more by culturing the electroporated population. In one embodiment, human T regulatory cells are expanded via anti-CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating immune cells can be found in U. S. Patent Numbers 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein in their entirety.

[0635] In one embodiment, the method of expanding the immune cells can further comprise isolating the expanded immune cells for further applications. In another embodiment, the method of expanding can further comprise a subsequent electroporation of the expanded immune cells followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded immune cells, transfecting the expanded immune cells, or electroporating the expanded immune cells with a nucleic acid, into the expanded population of immune cells, wherein the agent further stimulates the immune cell. The agent may stimulate the immune cells, such as by stimulating further expansion, effector function, or another immune cell function.

[0636] Methods

[0637] In one aspect, the present invention provides a method for killing an MSLN+cell. In another aspect, the present invention provides a method for killing a CEACAM5+cell. In another aspect, the present invention provides a method for killing an MSLN+ / CEACAM5+cell.

[0638] In one embodiment, a method for killing an MSLN+cell comprises contacting the cell with an MSLN CAR T cell, an MSLN CAR NK cell, an MSLN CAR mRNA, or an MSLN Attorney Docket No: 046483 -7482WO 1(04099)

[0639] expression construct, as described herein. In an embodiment, the MSLN+cell is a gastric cancer cell, a pancreatic cancer cell, a colorectal cancer cell, a glioma cell, a breast cancer cell, a liver cancer cell, or an ovarian cancer cell.

[0640] In another embodiment, a method for killing an CEACAM5+cell comprises contacting the cell with a CEACAM5 CAR T cell, a CEACAM5 CAR NK cell, a CEACAM5 CAR mRNA, or a CEACAM5 expression construct, as described herein. In an embodiment, the CEACAM5+ cell is a gastric cancer cell, a pancreatic cancer cell, a colorectal cancer cell, a glioma cell, a breast cancer cell, a liver cancer cell, or an ovarian cancer cell.

[0641] In another embodiment, a method for killing an MSLN+ / CEACAM5+cell comprises contacting the cell with an MSLN / CEACAM5 biCAR T ...

Claims

1. Attorney Docket No: 046483 -7482WO 1(04099)2.CLAIMS3.What is claimed:

1. A nanobody comprising the CDR1, CD2, and CDR3 amino acid sequences selected from the group consisting of:5.a. SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively,6.b. SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively,7.c. SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively,8.d. SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively,9.e. SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively,10.f. SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively,11.g. SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively,12.h. SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, and13.i. SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively,14.wherein the nanobody comprises an antigen binding domain that specifically binds mesothelin (MSLN) or CEACAM5.

2. The nanobody of claim 1, wherein the nanobody is a VHH comprising the amino acid sequence set forth in any one of SEQ ID NOs: 28-36 and / or encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 37-45.

3. The nanobody of claim 1 or 2, wherein the antigen binding domain specifically binds MSLN.

4. The nanobody of claim 1 or 2, wherein the antigen binding domain specifically binds CEACAM5.

5. A chimeric antigen receptor (CAR), comprising:19.the nanobody of any one of claims 1-4,20.a transmembrane domain, and21.an intracellular domain. Attorney Docket No: 046483 -7482WO 1(04099)6. The CAR of claim 5, wherein the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, and a transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, 0X40 (CD134), 4-1BB (CD137), and CD 154.

7. The CAR of claim 4, wherein the transmembrane domain comprises a transmembrane domain of CD8, optionally comprising the amino acid sequence of SEQ ID NO: 51.

8. The CAR of claim 4, wherein the transmembrane domain comprises a transmembrane domain of CD28, optionally comprising the amino acid sequence of SEQ ID NO: 52.

9. The CAR of any one of claims 5-8, wherein the CAR further comprises a hinge domain.

10. The CAR of claim 9, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an IgG4 hinge region, an artificial hinge domain, a CD8 hinge region, or any combination thereof.

11. The CAR of claim 10, wherein the hinge domain is an IgG4 hinge, optionally comprising the amino acid sequence of SEQ ID NO: 48.

12. The CAR of any one of claims 5-11, wherein the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

13. The CAR of claim 12, wherein the costimulatory signaling domain comprises a costimulatory signaling domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lek, TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, B7-H3 (CD276), a variant thereof, and a combination thereof.Attorney Docket No: 046483 -7482WO 1(04099)14. The CAR of claim 13, wherein the costimulatory signaling domain comprises a costimulatory signaling domain of 4- IBB, optionally comprising the amino acid sequence of SEQ ID NO: 55.

15. The CAR of claim 11, wherein the costimulatory signaling domain comprises a costimulatory signaling domain of CD28, optionally comprising the amino acid sequence of SEQ ID NO: 56.

16. The CAR of any one of claims 12-15, wherein the CAR comprises two costimulatory signaling domains.

17. The CAR of claim 16, wherein the costimulatory signaling domain comprises a CD28 costimulatory signaling domain and a 4- IBB costimulatory signaling domain, optionally wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 55 and the CD28 costimulatory domain comprises the amino acid sequence of SEQ ID NO: 56.

18. The CAR of any one of claims 12-17, wherein the intracellular signaling domain comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain (CD3Q, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

19. The CAR of claim 18, wherein the intracellular signaling domain comprises an intracellular domain of CD3(^, optionally wherein the intracellular domain of CD3 comprises the amino acid sequence of SEQ ID NO: 60.

20. The CAR of any one of claims 5-19, wherein the CAR comprises an anti-MSLN nanobody, an IgG4 hinge region, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, and a CD3ζ intracellular signaling domain.Attorney Docket No: 046483 -7482WO 1(04099)21. The CAR of claim 20, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 82-89.

22. The CAR of any one of claims 5-19, wherein the CAR comprises an anti-CEACAM5 nanobody, an IgG4 hinge region, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, and a CD3 intracellular signaling domain.

23. The CAR of claim 22, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 90 or 91.

24. The CAR of any one of claims 5-23, wherein the CAR comprises a second antigen binding domain.

25. The CAR of claim 24, wherein the second antigen binding domain is an antibody or an antigen-binding fragment thereof.

26. The CAR of claim 24 or 25, wherein the second antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a nanobody.

27. The CAR of any one of claims 24-26, wherein the second antigen-binding fragment is an scFv.

28. The CAR of any one of claims 24-26, wherein the second antigen-binding fragment is a nanobody.

29. The CAR of any one of claims 24-28, wherein the second antigen binding domain is a tumor-associated antigen (TAA) binding domain.

30. The CAR of claim 29, wherein the TAA binding domain specifically targets a TAA selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA,Attorney Docket No: 046483 -7482WO 1(04099)47.CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA / CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, R0R1, R0R2, TIM-3, TM4SF1, TnMucl, VEGFR2, variants thereof, and combinations thereof.

31. The CAR of any one of claims 24-30, wherein the CAR comprises an anti-MSLN binding domain and an anti-CEACAM5 binding domain.

32. The CAR of claim 31, wherein the anti-MSLN binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 28-35 and / or encodes the nucleic acid sequence of any one of SEQ ID NO: 37-44, respectively, and the anti-CEACAM5 binding domain comprises the amino acid sequence of SEQ ID NO: 36 and / or encodes the nucleotide sequence of SEQ ID NO: 45.

33. The CAR of any one of claims 24-32, wherein the CAR comprises a self-cleaving 2A peptide domain selected from the group consisting of T2A, P2A, E2A and F2A.

34. The CAR of any one of claims 24-33, wherein the CAR is a Split Bi-CAR.

35. The CAR of claim 34, wherein the Split Bi-CAR comprises from its amino to carboxy terminus, the second antigen binding domain, a first costimulatory signaling domain, an intracellular signaling domain, the anti-MSLN nanobody, and a second costimulatory signaling domain.

36. The CAR of claim 34, wherein the Split Bi-CAR comprises from its amino to carboxy terminus, the anti-MSLN nanobody, a first costimulatory signaling domain, an intracellularAttorney Docket No: 046483 -7482WO 1(04099)54.signaling domain, the second antigen binding domain, and a second costimulatory signaling domain.

37. The CAR of claim 34, wherein the Split Bi-CAR comprises from its amino to carboxy terminus, the anti-CEACAM5 nanobody, a first costimulatory domain, an intracellular signaling domain, the second antigen binding domain, and a second costimulatory domain.

38. The CAR of claim 34, wherein the Split Bi-CAR comprises from its amino to carboxy terminus, the second antigen binding domain, a first costimulatory domain, an intracellular signaling domain, the anti-CEACAM5 nanobody, and a second costimulatory domain.

39. The CAR of any one of claims 35-38, wherein one costimulatory signaling domain is a CD28 costimulatory signaling domain and another costimulatory signaling domain is a 4-1BB costimulatory signaling domain.

40. The CAR of claim 35, wherein the Split Bi-CAR comprises from its amino to carboxy terminus, an anti-CEACAM5 nanobody, a CD28 costimulatory signaling domain, a CD3q intracellular signaling domain, the anti-MSLN nanobody, and a 4- IBB costimulatory signaling domain.

41. The CAR of claim 40, wherein the Split Bi-CAR comprises the amino acid sequence of SEQ ID NO: 110 or 111.

42. The CAR of any one of claims 24-33, wherein the CAR is a Tandem Bi-CAR.

43. The CAR of claim 42, wherein the Tandem Bi-CAR comprises, from its amino to carboxy terminus, the anti-MSLN nanobody, the second antigen binding domain, a first costimulatory signaling domain, a second costimulatory signaling domain, and an intracellular signaling domain.

44. The CAR of claim 42, wherein the Tandem Bi-CAR comprises from its amino to carboxyAttorney Docket No: 046483 -7482WO 1(04099)63.terminus, the second antigen binding domain, the anti-MSLN nanobody, a first costimulatory signaling domain, a second costimulatory signaling domain, and an intracellular signaling domain.

45. The CAR of claim 42, wherein the Tandem Bi-CAR comprises, from its amino to carboxy terminus, the anti-CEACAM5 nanobody, the second antigen binding domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3(^ intracellular signaling domain.

46. The CAR of claim 42, wherein the Tandem Bi-CAR comprises, from its amino to carboxy terminus, the second antigen binding domain, the anti-CEACAM5 nanobody, a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain.

47. The CAR of any one of claims 43-46, wherein one costimulatory signaling domain is a CD28 costimulatory signaling domain and another costimulatory signaling domain is a 4-1BB costimulatory signaling domain.

48. The CAR of claim 44, wherein the Tandem Bi-CAR comprises from its amino to carboxy terminus, the anti-CEACAM5 nanobody, an anti-MSLN nanobody, a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, and a CD3 intracellular signaling domain.

49. The CAR of claim 48, wherein the Tandem Bi-CAR comprises the amino acid sequence of SEQ ID NO: 114 or 115.

50. A nucleic acid encoding the nanobody of any one of claims 1-4.

51. A nucleic acid encoding the CAR of any one of claims 5-23.

52. A nucleic acid encoding the CAR of any one of claims 24-49.Attorney Docket No: 046483 -7482WO 1(04099)53. The nucleic acid of claim 50 or 51, wherein the nucleic acid encodes a CAR comprising the anti-MSLN nanobody, a transmembrane domain, and an intracellular domain.

54. The nucleic acid of claim 50 or 51, wherein the nucleic acid encodes a CAR comprising the anti-CEACAM5 nanobody, a transmembrane domain, and an intracellular domain.

55. The nucleic acid of any one of claims 53 or 54, wherein the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, and a transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, 0X40 (CD134), 4-1BB (CD137), and CD154.

56. The nucleic acid of claim 55, wherein the transmembrane domain comprises a transmembrane domain of CD8, optionally wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 65 and / or encodes the amino acid sequence of SEQ ID NO: 51.

57. The nucleic acid of claim 55, wherein the transmembrane domain comprises a transmembrane domain of CD28, optionally wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 66 and / or encodes the amino acid sequence of SEQ ID NO: 52.

58. The nucleic acid of any one of claims 50-57, wherein the CAR further comprises a hinge domain.

59. The nucleic acid of claim 58, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an IgG4 hinge region, an artificial hinge domain, a CD8 hinge region, or any combination thereof.

60. The nucleic acid of claim 58, wherein the hinge domain is an IgG4 hinge, optionally comprising the nucleotide sequence of SEQ ID NO: 62 and / or encoding the amino acid sequenceAttorney Docket No: 046483 -7482WO 1(04099)80.of SEQ ID NO: 48.

61. The nucleic acid of any one of claims 53-60, wherein the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

62. The nucleic acid of claim 51, wherein the costimulatory signaling domain comprises a costimulatory signaling domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lek, TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, B7-H3 (CD276), a variant thereof, and a combination thereof.

63. The nucleic acid of claim 62, wherein the costimulatory signaling domain comprises a costimulatory signaling domain of 4- IBB, optionally wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 69 and / or encodes the amino acid sequence of SEQ ID NO: 55.

64. The nucleic acid of claim 62, wherein the costimulatory signaling domain comprises a costimulatory signaling domain of CD28, optionally wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 70 and / or encoding the amino acid sequence of SEQ ID NO: 56.

65. The nucleic acid of any one of claims 62-64, wherein the CAR comprises two costimulatory signaling domains.

66. The nucleic acid of claim 65, wherein the CAR comprises a CD28 costimulatory signaling domain and a 4- IBB costimulatory signaling domain, optionally wherein the nucleic acid encoding the CD28 costimulatory signaling domain comprises the nucleotide sequence of SEQ ID NO: 70 or encodes the amino acid sequence of SEQ ID NO: 56 and / or optionally wherein the nucleic acid encoding the 4-1BB costimulatory signaling domain comprises the nucleotide sequence of SEQ ID NO: 69 or encodes the amino acid sequence of SEQ ID NO: 55.Attorney Docket No: 046483 -7482WO 1(04099)67. The nucleic acid of any one of claims 61-66, wherein the intracellular signaling domain comprises an intracellular domain selected from the group consisting cytoplasmic signaling domains of a human CD3 zeta chain (CD3Q, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

68. The nucleic acid of claim 67, wherein the intracellular signaling domain comprises an intracellular domain of CD3, optionally wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 73 or 74, or encodes the amino acid sequence of SEQ ID NO: 59 or 60.

69. The nucleic acid of claim 53, wherein the CAR comprises the anti-MSLN nanobody, an IgG4 hinge region, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, a 4- IBB costimulatory signaling domain, and a CD3ζ intracellular signaling domain.

70. The nucleic acid of claim 69, wherein the CAR is encoded by the nucleotide sequence of any one of SEQ ID NOs: 94-101.

71. The nucleic acid of claim 54, wherein the CAR comprises the anti-CEACAM5 nanobody, an IgG4 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain.

72. The nucleic acid of claim 71, wherein the CAR comprises the nucleotide sequence of SEQ ID NO: 102 or 103, and / or encodes the amino acid sequence of SEQ ID NO: 90 or 91.

73. The nucleic acid of any one of claims 52-72, wherein the CAR comprises a second antigen binding domain.

74. The nucleic acid of claim 73, wherein the second antigen binding domain is an antibody or an antigen-binding fragment thereof.Attorney Docket No: 046483 -7482WO 1(04099)75. The nucleic acid of claim 73 or 74, wherein the second antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a nanobody.

76. The nucleic acid of any one of claims 73-75, wherein the second antigen-binding fragment is an scFv.

77. The nucleic acid of any one of claims 73-75, wherein the second antigen-binding fragment is a nanobody.

78. The nucleic acid of any one of claims 73-77, wherein the second antigen binding domain is a tumor-associated antigen (TAA) binding domain.

79. The nucleic acid of claim 78, wherein the TAA binding domain specifically targets a TAA selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA, CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD 117, CD 123, CD 133, CD 147, CD 171, CD276, CEA / CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, variants thereof, and combinations thereof.

80. The nucleic acid of any one of claims 73-79, wherein the nucleic acid encodes a CAR comprising an anti-MSLN binding domain and an anti-CEACAM5 binding domain.

81. The nucleic acid of claim 80, wherein the anti-MSLN binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 28-35 and / or encodes the nucleic acid sequence of any one of SEQ ID NO: 37-44, respectively, and the anti-CEACAM5 binding domain comprises the amino acid sequence of SEQ ID NO: 36 and / or encodes the nucleotide sequence of SEQ IDAttorney Docket No: 046483 -7482WO 1(04099)102.NO: 45.

82. The nucleic acid of any one of claims 73-81, wherein the CAR comprises a self-cleaving 2A peptide domain selected from the group consisting of T2A, P2A, E2A and F2A, optionally wherein the self-cleaving 2A peptide domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 160-163.

83. The nucleic acid of any one of claims 73-82, wherein the CAR is a Split Bi-CAR.

84. The nucleic acid of claim 83, wherein the Split Bi-CAR comprises from its amino to carboxy terminus, the second antigen binding domain, a first costimulatory signaling domain, an intracellular signaling domain, the anti-MSLN nanobody, and a second costimulatory signaling domain.

85. The nucleic acid of claim 83, wherein the Split Bi-CAR comprises from its amino to carboxy terminus, the anti-MSLN nanobody, a first costimulatory signaling domain, an intracellular signaling domain, the second antigen binding domain, and a second costimulatory signaling domain.

86. The nucleic acid of claim 83, wherein the Split Bi-CAR comprises from its amino to carboxy terminus, the anti-CEACAM5 nanobody, a first costimulatory domain, an intracellular signaling domain, the second antigen binding domain, and a second costimulatory domain.

87. The nucleic acid of claim 83, wherein the Split Bi-CAR comprises from its amino to carboxy terminus, the second antigen binding domain, a first costimulatory domain, an intracellular signaling domain, the anti-CEACAM5 nanobody, and a second costimulatory domain.

88. The nucleic acid of any one of claims 84-87, wherein one costimulatory signaling domain is a CD28 costimulatory signaling domain and another costimulatory signaling domain is a 4-1BB costimulatory signaling domain.Attorney Docket No: 046483 -7482WO 1(04099)89. The nucleic acid of claim 83 or 84, wherein the Split Bi-CAR comprises from its amino to carboxy terminus, an anti-CEACAM5 nanobody, a CD28 costimulatory signaling domain, a CD3ζ intracellular signaling domain, the anti-MSLN nanobody, and a 4- IBB costimulatory signaling domain.

90. The nucleic acid of claim 89, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 117 or 119 and / or encodes the amino acid sequence of SEQ ID NO: 110 or 111.

91. The nucleic acid of any one of claims 73-82, wherein the CAR is a Tandem Bi-CAR.

92. The nucleic acid of claim 91, wherein the Tandem B-CAR comprises, from its amino to carboxy terminus, the anti-MSLN nanobody, the second antigen binding domain, a first costimulatory signaling domain, a second costimulatory signaling domain, and an intracellular signaling domain.

93. The nucleic acid of claim 91, wherein the Tandem Bi-CAR comprises from its amino to carboxy terminus, the second antigen binding domain, the anti-MSLN nanobody, a first costimulatory signaling domain, a second costimulatory signaling domain, and an intracellular signaling domain.

94. The nucleic acid of claim 91, wherein the Tandem Bi-CAR comprises, from its amino to carboxy terminus, the anti-CEACAM5 nanobody, the second antigen binding domain, a CD28 costimulatory domain, a 4- IBB costimulatory domain, and a CD3ζ intracellular signaling domain.

95. The nucleic acid of claim 91, wherein the Tandem Bi-CAR comprises, from its amino to carboxy terminus, the second antigen binding domain, the anti-CEACAM5 nanobody, a CD28 costimulatory domain, a 4- IBB costimulatory domain, and a CD3ζ intracellular signaling domain.

96. The nucleic acid of any one of claims 92-95, wherein one costimulatory signaling domain isAttorney Docket No: 046483 -7482WO 1(04099)118.a CD28 costimulatory signaling domain and another costimulatory signaling domain is a 4- IBB costimulatory signaling domain.

97. The nucleic acid of claim 91, wherein the Tandem Bi-CAR comprises, from its amino to carboxy terminus, an anti-CEACAM5 nanobody, the anti-MSLN nanobody, a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, and a CD3 intracellular signaling domain.

98. The nucleic acid of claim 97, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 122 or 123, and / or encodes the amino acid sequence of SEQ ID NO: 114 or 115.

99. The nucleic acid of any one of claims 50-98, wherein the nucleic acid is an mRNA.

100. A vector comprising the nucleic acid of any one of claims 50-99.

101. The vector of claim 100, wherein the vector is an expression vector.

102. The vector of claim 100 or 101, wherein the vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

103. The vector of claim 102, wherein the vector is a lentiviral vector.

104. The vector of claim 103, wherein the lentiviral vector is a self-inactivating HIV vector comprising an LTR comprising a U3 deletion.

105. The vector of claim 104, wherein the HIV vector comprises a partial gag sequence, optionally wherein the partial gag sequence comprises the nucleotide sequence of SEQ ID NO: 133.

106. The vector of claim 104 or 105, wherein the HIV vector further comprises a central polypurine tract (cPPT), optionally wherein the cPPT comprises the nucleotide sequence of SEQAttorney Docket No: 046483 -7482WO 1(04099)129.ID NO: 134.

107. The vector of any one of claims 99-106, further comprising a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE), optionally wherein the WPRE comprises the nucleotide sequence of SEQ ID NO: 135.

108. The vector of any one of claims 100-107, wherein the vector comprises the nucleic acid of any one of claims 50-72.

109. The vector of any one of claims 100-107, wherein the vector comprises the nucleic acid of any one of claims 73-99.

110. The vector of claim 109, comprising the nucleotide sequence of any one of SEQ ID NOs: 124-129.

111. A cell comprising the CAR of any one of claims 5-49, the nucleic acid of any one of claims 50-99, or the vector of any one of claims 100-110.

112. The cell of claim 111, wherein the cell is an immune cell or precursor cell thereof.

113. The cell of claim 112, wherein the cell is a natural killer (NK) cell or a T cell.

114. The cell of claim 112, wherein the cell is a gamma delta T cell.

115. The cell of any one of claims 111-114, wherein the cell is an autologous cell.

116. The cell of any one of claims 111-114, wherein the cell is an allogeneic cell.

117. The cell of any one of claims 111-116, wherein the cell comprises the CAR of any one of claims 5-23, the nucleic acid of any one of claims 50-72 or 99, or the vector of any one of claims 100-108, wherein the cell expresses a single exogenous antigen binding domain.Attorney Docket No: 046483 -7482WO 1(04099)118. The cell of any one of claims 109-114, wherein the cell comprises the CAR of any one of claims 24-49, the nucleic acid of any one of claims 73-99, or the vector of any one of claims 100-110, wherein the cell expresses two exogenous antigen binding domains.

119. A pharmaceutical composition comprising the anti-MSLN or anti-CEACAM5 nanobody of any one of claims 1-4, the CAR of any one of claims 5-49, the nucleic acid of any one of claims 50-99, or the cell of any one of claims 111-118; and a pharmaceutically acceptable carrier.

120. A method for killing a cell, comprising contacting the cell with the cell of claim 117 or 118, wherein the cell is an MSLN+and / or CEACAM5+cell.

121. The method of claim 120, wherein the cell is an MSLN+cell.

122. The method of claim 120, wherein the cell is a CEACAM5+cell.

123. The method of claim 120, wherein the cell is an MSLN+CEACAM5+cell.

124. The method of any one of claims 120-123, wherein the cell is a gastric cancer cell, a pancreatic cancer cell, a colorectal cancer cell, a glioma cell, a breast cancer cell, a liver cancer cell, or an ovarian cancer cell.

125. The method of claim 123 or 124, wherein the MSLN+ / CEACAM5+cell is contacted with a cell comprising the nucleic acid of claim 90 or 98.

126. A method for treating cancer in a subject in need thereof, comprising: administering an effective amount of the pharmaceutical composition of claim 119 to the subject, thereby treating the cancer.

127. The method of claim 126, wherein the cancer is a solid tumor.Attorney Docket No: 046483 -7482WO 1(04099)128. The method of claim 127, wherein the solid tumor is selected from the group consisting of gastric cancer, pancreatic cancer, colorectal cancer, melanoma, glioma, breast cancer, liver cancer, lung cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, hepatocellular carcinoma, rectal cancer, kidney cancer, prostate cancer, bronchus cancer, urinary bladder cancer, and brain cancer.

129. The method of claim 127 or 128, wherein the solid tumor is gastric cancer, pancreatic cancer, or colorectal cancer.

130. The method of any one of claims 126-129, wherein the pharmaceutical composition is administered intratum orally.

131. The method of claim 126, wherein the cancer is a hematologic cancer.

132. The method of claim 131, wherein the hematologic cancer is a myeloid malignancy selected from the group consisting of acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), myelodysplastic neoplasm, and myeloproliferative neoplasm.

133. A method for preparing the cell of any one of claims 111-118, comprising: introducing into the cell the nucleic acid of any one of claims 50-99, or the vector of any one of claims 100-110.

134. An antibody-drug conjugate, comprising:158.the nanobody of any one of claims 1-4; and159.a drug, toxin, or radioisotope conjugated to the nanobody, wherein the nanobody specifically binds MSLN or CEACAM5.

135. The antibody-drug conjugate of claim 134, wherein the nanobody specifically binds MSLN.

136. The antibody-drug conjugate of claim 134, wherein the nanobody specifically binds CEACAM5.Attorney Docket No: 046483 -7482WO 1(04099)137. The antibody-drug conjugate of any one of claims 134-136, wherein maytansinoid (DM1), SSTR2 -binding octreotide, paclitaxel, auristatin, MMAE, MMAF, dauxrubicin, duocarmycin A, 5-fluoruracil, methotrexate, tubulin polymerization inhibitors, ravtansine (DM4), Ricin A, 90Y, 177Lu, or 11 Hn is conjugated to the nanobody.

138. A bispecific antibody comprising the anti-MSLN nanobody of claim 3 and the anti-CEACAM5 nanobody of claim 4.

139. The bispecific antibody of claim 138, further comprising one or more immunoglobulin constant regions.

140. A method for treating cancer in a subject in need thereof, comprising: administering an effective amount of the antibody-drug conjugate of any one of claims 134-137 to the subject, thereby treating the cancer.

141. A method for treating cancer in a subject in need thereof, comprising: administering an effective amount of the bispecific antibody of claim 138 or 139 to the subject, thereby treating the cancer.

142. A method for treating cancer in a subject in need thereof, comprising: administering an effective amount of the nanobody of any one of claims 1-4 to the subject, thereby treating the cancer.

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