阅读说明：本技术 Lym-1和lym-2抗体组合物及改进的car构建体 (LYM-1 and LYM-2 antibody compositions and improved CAR constructs ) 是由 艾伦·L·爱泼斯坦 胡培生 郑龙 于 2020-02-14 设计创作，主要内容包括：本文提供了新的抗体和包含这些抗体的抗原结合结构域的嵌合抗原受体(CAR)细胞。还提供了包含所述抗体和细胞、编码所述抗体和CAR的载体或质粒的组合物,以及产生它们的方法或使用它们检测或治疗癌症的方法,以及用于实施所述方法的试剂盒。(Provided herein are novel antibodies and Chimeric Antigen Receptor (CAR) cells comprising the antigen binding domains of these antibodies. Also provided are compositions comprising the antibodies and cells, vectors or plasmids encoding the antibodies and CARs, and methods of producing them or methods of using them to detect or treat cancer, as well as kits for practicing the methods.)

1. An antibody, comprising:

(i) a Heavy Chain (HC) immunoglobulin variable domain sequence comprising SEQ ID NO: 2 or 6 or an equivalent of each thereof; and/or

(ii) A Light Chain (LC) immunoglobulin variable domain sequence comprising SEQ ID NO: 4 or 8 or an equivalent of each thereof.

2. The antibody of claim 1, wherein the Heavy Chain (HC) immunoglobulin variable domain sequence comprises the amino acid sequence of SEQ ID NO: 2, and the Light Chain (LC) immunoglobulin variable domain sequence comprises the amino acid sequence of SEQ ID NO: 4, or an equivalent of each thereof.

3. The antibody of claim 1, wherein the Heavy Chain (HC) immunoglobulin variable domain sequence comprises the amino acid sequence of SEQ ID NO: 2, and the Light Chain (LC) immunoglobulin variable domain sequence comprises the amino acid sequence of SEQ ID NO: 8, or an equivalent of each thereof.

4. The antibody of claim 1, wherein the Heavy Chain (HC) immunoglobulin variable domain sequence comprises the amino acid sequence of SEQ ID NO: 6, and the Light Chain (LC) immunoglobulin variable domain sequence comprises the amino acid sequence of SEQ ID NO: 4, or an equivalent of each thereof.

5. The antibody of claim 1, wherein the Heavy Chain (HC) immunoglobulin variable domain sequence comprises the amino acid sequence of SEQ ID NO: 6, and the Light Chain (LC) immunoglobulin variable domain sequence comprises the amino acid sequence of SEQ ID NO: 8, or an equivalent of each thereof.

6. An antibody, comprising:

(i) a Heavy Chain (HC) immunoglobulin variable domain sequence comprising SEQ ID NO: 10 or an equivalent thereof; and/or

(ii) A Light Chain (LC) immunoglobulin variable domain sequence comprising SEQ ID NO: 12 or an equivalent thereof.

7. An antigen-binding fragment of the antibody of any one of claims 1 to 6.

8. A polypeptide comprising SEQ ID NO: 2. 4, 6, 8, 10 or 12, or an equivalent of each thereof.

9. A Chimeric Antigen Receptor (CAR) comprising: (a) the antigen binding domain of the antibody of any one of claims 1 to 6, (b) a hinge domain, (c) a transmembrane domain, and (d) an intracellular signaling domain or a DAP domain.

10. The CAR of claim 9, further comprising one or more co-stimulatory signaling regions.

11. The CAR of claim 9 or 10, wherein the DAP domain is DAP10 and/or DAP 12.

12. The CAR of any one of claims 9 to 11, further comprising peptide AVPPQQWALS inserted after the HC and LC variable domains.

13. An isolated nucleic acid sequence, wherein the nucleic acid sequence comprises a sequence selected from any of the sequences disclosed herein or an equivalent thereof, and optionally is operably linked to a promoter and/or enhancer element.

14. An isolated nucleic acid sequence encoding the antibody of any one of claims 1 to 6, the antigen-binding fragment of claim 7, the polypeptide of claim 8, or the CAR of any one of claims 9 to 12.

15. A vector comprising the isolated nucleic acid sequence of claim 14.

16. An isolated cell comprising the antibody of any one of claims 1 to 6, the antigen-binding fragment of claim 7, the polypeptide of claim 8, the CAR of any one of claims 9 to 12, the isolated nucleic acid of claim 13 or 14, and/or the vector of claim 15.

17. The isolated cell of claim 16, wherein the isolated cell is an immune cell.

18. The isolated cell of claim 17, wherein the immune cell is a T cell or a Natural Killer (NK) cell.

19. A composition comprising a vector and one or more of the antibody of any one of claims 1 to 6, the antigen-binding fragment of claim 7, the polypeptide of claim 8, the CAR of any one of claims 9 to 12, the isolated nucleic acid of claim 13 or 14, the vector of claim 15, and/or the isolated cell of any one of claims 16 to 18.

20. A method of generating a cell expressing a CAR, comprising:

(i) introducing a nucleic acid sequence encoding the CAR of any one of claims 9 to 12 into a population of immune cells; and

(ii) (ii) selecting a subpopulation of immune cells that have been successfully transduced with the nucleic acid sequence of step (i), thereby generating cells expressing the CAR.

21. The method of claim 20, wherein the immune cell is a T cell or an NK cell.

22. The method of claim 20 or 21, wherein the population of immune cells has been modified to reduce or eliminate expression of endogenous immune cell receptors.

23. A method of inhibiting tumor growth and/or treating cancer and/or preventing cancer recurrence in a subject in need thereof, comprising administering to the subject an effective amount of the antibody of any one of claims 1 to 6, the antigen binding domain of claim 7, the polypeptide of claim 8, or the CAR of any one of claims 9 to 12.

24. The method of claim 23, further comprising administering one or more anti-cancer therapeutics, checkpoint inhibitors, regulatory T cells (tregs), Myeloid Derived Suppressor Cells (MDSCs), fluorouracil (5-FU), Histone Deacetylase (HDAC) inhibitors, IL-12 therapy, CpG (TLR 9 agonists), and/or interferon gene Stimulator (STING) pathway agonists.

25. The method of claim 23 or 24, wherein the tumor or cancer cell expresses or overexpresses Lym1 and/or Lym2 or CD 19.

26. A method of inhibiting proliferation of a cancer cell or cancer stem cell, comprising contacting the cell with an effective amount of the antibody of any one of claims 1 to 6, the antigen binding domain of claim 7, the polypeptide of claim 8, or the CAR of any one of claims 9 to 12.

27. A method for determining whether a subject is likely to respond or unlikely to respond to a therapy, comprising contacting a sample isolated from a patient with the antibody of any one of claims 1 to 6 or the antigen-binding fragment of claim 7, the polypeptide of claim 8, wherein the presence of a complex between the antibody, antigen-binding fragment, or polypeptide indicates that the subject is likely to respond to the therapy and the absence of the complex indicates that the subject is unlikely to respond to the therapy.

28. A method for monitoring therapy in a subject, comprising contacting a sample isolated from the subject with one or more of the antibody of any one of claims 1 to 6, the antigen-binding fragment of claim 7, the polypeptide of claim 8, or the polypeptide of claim 8, and detecting the complex in the sample.

29. A kit comprising one or more of the antibody of any one of claims 1 to 6, the antigen-binding fragment of claim 7, the polypeptide of claim 8, or the CAR of any one of claims 9 to 12, and instructions for use.

Background

The present disclosure relates generally to the field of human immunology, and in particular to cancer immunotherapy.

The following discussion of the background art is intended to aid the reader in understanding the present disclosure and is not an admission that it describes or constitutes prior art against the present disclosure. Throughout the disclosure of this invention, various publications are referenced by Arabic numerals, a full bibliographic citation of which precedes the claims. These references, as well as the cited techniques and patent documents, are incorporated herein by reference.

Lym-1 and Lym-2 are directed against MHC class II HLA-DR molecules known to be expressed predominantly on the surface of human B cells, dendritic cells and B cell derived lymphomas and leukemias.

Murine monoclonal antibodies Lym-1 and Lym-2 (Epstein AL, et AL, 1987, Cancer Res. 47: 830-840) were originally developed for targeting HLA-Dr antigens expressed on the cell surface of most human B-cell malignancies (Rose et AL, 1996, Cancer Immunol Immunother 43(1): 26-30). Lym-1 binding epitopes were then identified, which are discontinuous sites on the human HLA-Dr light chain. Lym-2 does not compete with Lym-1 and therefore binds to a different epitope on the HLA-Dr antigen. Lym-1 has been tested in humans as an I-131 radiolabeled drug and as a naked Antibody for the treatment of diffuse large cell lymphoma and has proven to be effective and safe (DeNardo SJ et al, 1988, antibodies, Immunoconjugates, and Radiopharmaceuticals 1: 17-33; DeNardo, S.J. et al, 1988, int. J. Cancer 3: 96-101; and Hu E. et al, 1989, histological Oncology 7: 155-. Unlike antibodies to other B cell antigens (e.g., CD19 and CD 20), Lym-1, but not Lym-2, undergoes tumor cell apoptosis upon binding due to the antigen being located in lipid rafts on the surface of human lymphoma cells (Epstein AL, et AL, 1987, Cancer Res. 47: 830-840). Furthermore, Lym-1 and Lym-2 antigens are not shed or internalized to any extent and are only expressed on normal circulating and lymph node-bound B cells at concentrations of 1/4 compared to lymphoma cells.

Summary of The Invention

Due to the above properties, Lym-1 and Lym-2 are ideal targeting antibodies useful for the treatment of antigen positive leukemias and lymphomas. Recently, both Lym-1 and Lym-2 have been used to generate potent human CAR T cells as novel compositions of matter for immunotherapy of these malignancies and have been patented. The disclosure shows that the newly generated humanized Lym-1 and Lym-2 have lower affinity for antigen than the parent antibody, can be used to generate clinically effective human CAR T cells to treat B cell malignancies, and are effective against metastatic antigen positive tumors upon NSG mouse xenograft. These data provide key information on the potential use of these humanized antibody constructs in generating human CAR T cells capable of treating B cell tumor patients.

Since recent, unprecedented results have been obtained in the treatment of B-cell lymphomas and leukemias using autologous therapy with genetically engineered Chimeric Antigen Receptor (CAR) T cells, many laboratories have begun to apply this approach to solid tumors. CAR-modified cells combine the HLA-independent targeting specificity of monoclonal antibodies with the cytolytic activity, proliferation and homing properties of activated T cells, but do not respond to checkpoint inhibition. Because they are able to kill antigen-expressing targets directly, CAR cells are highly toxic to any antigen-positive cell or tissue, and it is therefore desirable to construct CARs with highly specific antibodies. In one aspect, disclosed herein are novel antibodies, CARs, and methods of their use in diagnosis and therapy, having desirable safety and efficacy characteristics.

In one aspect, the disclosure provides an antibody comprising, consisting essentially of, or consisting of: a Heavy Chain (HC) immunoglobulin variable domain sequence comprising SEQ ID NO: 2 or 6 or an equivalent of each thereof; and/or a Light Chain (LC) immunoglobulin variable domain sequence comprising SEQ ID NO: 4 or 8 or an equivalent of each thereof. In another aspect, disclosed herein is an antibody comprising, consisting essentially of, or consisting of the sequence of seq id no: comprises the amino acid sequence of SEQ ID NO: 10 or an equivalent thereof; and/or comprises SEQ ID NO: 12 or an equivalent thereof, or a Light Chain (LC) immunoglobulin variable domain sequence.

In one aspect, the antibodies disclosed herein are IgA, IgD, IgE, IgG, or IgM antibodies. The constant region of the antibody may also vary. For example, antibodies having Fc regions of any isotype can be provided: IgA (IgA 1, IgA 2), IgD, IgE, IgG (IgG 1, IgG2, IgG3, IgG 4) or IgM. In one embodiment, the constant region of an antibody disclosed herein is an IgGl constant region or an Ig κ constant region. In a particular aspect, an antibody disclosed herein can further comprise, consist essentially of, or consist of a detectable label or a purification label. Further provided herein is a method of producing an antibody disclosed herein, comprising, consisting essentially of, or consisting of: culturing the isolated cell described above, wherein the isolated cell is optionally a mammalian cell.

Further provided herein are antigen binding fragments of the antibodies disclosed herein. In some aspects of the antibodies provided herein, the antibody fragment is selected from Fab, F (ab) '2, Fab', scFv, and Fv. In another aspect, provided herein are isolated antibodies or fragments thereof as disclosed herein and a detectable or purification label, alone or in combination with an antigen or fragment thereof. Further provided herein are ex vivo (ex vivo) cells comprising, consisting essentially of, or consisting of the antigen/antibody complexes.

The disclosure provides polypeptides comprising SEQ ID NO: 2. 4, 6, 8, 10 or 12 or an equivalent of each thereof, or consists essentially of, or consists of. Further provided is an isolated polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of an antibody disclosed herein or a fragment thereof and an isolated polynucleotide encoding the same.

Also further provided are isolated nucleic acids encoding the antibodies and fragments thereof disclosed herein. In one aspect, the isolated nucleic acid sequence comprises SEQ ID NO: 1. 3, 5, 7, 9 and 11 or their respective equivalents, or consist essentially of, or consist of. In one aspect, the polynucleotide sequence is operably linked to a promoter and/or enhancer element. They may also be used in combination with a vector or a suitable host cell and/or a suitable vector for diagnostic or therapeutic use. In one aspect, the nucleic acid is contained in a host cell for recombinant production of polypeptides and proteins. The host cell may be a eukaryotic cell or a prokaryotic cell.

Aspects disclosed herein relate to a Chimeric Antigen Receptor (CAR) comprising, consisting essentially of, or consisting of: (a) an antigen binding domain of any of the antibodies disclosed herein, (b) a hinge domain, (c) a transmembrane domain, and (d) an intracellular signaling domain. In one aspect, the CAR further comprises, consists essentially of, or consists of one or more co-stimulatory signaling regions. In another aspect, a CAR disclosed herein is a first generation CAR, a second generation CAR, a third generation CAR, or a fourth generation CAR.

Also provided herein is a Chimeric Antigen Receptor (CAR) comprising, consisting essentially of, or consisting of the sequence of seq id no: (a) an antigen binding domain (e.g., of an anti-Lym antibody), (b) a hinge domain, (c) a transmembrane domain, and (d) a DAP 10 and/or DAP 12 domain. In another aspect, the CAR further comprises the 10 amino acid epitope "AVPPQQWALS" inserted directly after at least the antigen binding domain. The antigen binding domain may be from any suitable species, such as mammalian, murine, rat or e.g. human.

The disclosure herein provides a CAR construct comprising, or consisting essentially of, or consisting of: (a) an antigen binding domain of an antibody (e.g., an anti-Lym antibody, a humanized anti-Lym antibody, or an anti-CD 19 antibody), (b) a hinge domain, (c) a transmembrane domain, and (d) an intracellular signaling domain comprising one or more transmembrane domains of DAP10 and/or DAP 12. Non-limiting examples of antibodies include anti-Lym-1, anti-huLym-1 or Lym2, anti-Lym 2 antibodies, or anti-CD 19 antibodies, which may be from any suitable species. The construct may further comprise, consist essentially of, or consist of one or more linker polypeptides linking the above elements (a) to (d). In another aspect, the CAR further comprises, consists essentially of, or consists of a leader peptide located at the amine terminus of the antigen binding domain. In one embodiment, the hinge domain is a CD8 a or IgG1 hinge domain. In another aspect, the transmembrane domain comprises a CD8 a transmembrane domain. In another aspect, the CAR further comprises the 10 amino acid epitope "AVPPQQWALS" inserted directly after at least the antigen binding domain. The antigen binding domain may be from any suitable species, such as mammalian, murine, rat or e.g. human.

In particular embodiments, the CAR comprises, or consists essentially of, or consists of the following sequence: the antigen binding domain of any of the antibodies or fragments thereof disclosed herein (e.g., scFv), the CD8 a hinge domain or the IgG1 hinge domain, the CD8 a transmembrane domain, and the DAP 10 and/or DAP 12 transmembrane domain. In another aspect, the CAR includes DAP 10 and DAP 12 transmembrane domains.

In a particular aspect, the antibodies or CARs of the present disclosure may further comprise, consist essentially of, or consist of a detectable label or a purification label.

In another embodiment, the disclosed CAR comprises, or consists essentially of, or consists of the following sequence: a CD8 a or IgGl hinge domain, the transmembrane domain comprising a CD28 or CD8 a transmembrane domain, the one or more co-stimulatory signaling domains being selected from the group consisting of CD27, CD28, 4-IBB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD27, LIGHT, NKG2C, and B7-H3; and the intracellular signaling domain comprises a CD3 zeta signaling domain.

Also provided herein is a method of making a cell expressing a CAR (e.g., an anti-Lym CAR or anti-CD 19), comprising, consisting essentially of, or consisting of the steps of: introducing a nucleic acid sequence encoding a CAR disclosed herein into a population of immune cells; and (ii) selecting a subpopulation of immune cells that have been successfully transduced with the nucleic acid sequence of step (i), thereby generating cells expressing the CAR.

Aspects of the disclosure relate to an isolated cell comprising, consisting essentially of, or consisting of a CAR of the disclosure, and methods of producing such cells.

Also provided herein is a vector comprising, consisting essentially of, or consisting of an isolated nucleic acid sequence encoding an antibody or CAR disclosed herein. In one aspect, the disclosure provides a vector comprising, consisting essentially of, or consisting of an isolated nucleic acid sequence encoding the antibody or CAR construct.

In another aspect, the present disclosure provides a composition comprising, consisting essentially of, or consisting of a carrier (carrier) and one or more of an antibody, antigen-binding fragment, polypeptide, CAR, isolated nucleic acid, vector (vector), and/or isolated cell disclosed herein.

Other method aspects disclosed herein relate to a method of inhibiting tumor growth and/or treating cancer and/or preventing recurrence of cancer in a subject in need thereof, comprising, consisting essentially of, or consisting of: administering to the subject an effective amount of a CAR (e.g., anti-Lym or CD 19) expressing cell provided herein, an effective amount of an antibody, an effective amount of an antigen binding fragment thereof, and/or an effective amount of a polypeptide provided herein. In one aspect, the CAR expresses an antigen binding domain directed against an antigen expressed on a cancer or tumor cell to be treated or inhibited. In one aspect, the CAR-expressing cells are autologous or allogeneic to the subject, and optionally are for first-line, second-line, third-line, fourth-line, or fifth-line therapy. In another aspect, the tumor or cancer cell expresses or overexpresses a CAR-binding antigen, e.g., CD19, Lym1, and/or Lym 2. In another aspect, the cancer or tumor is selected from a carcinoma, sarcoma, or leukemia. In a particular aspect, the tumor or cancer is a B cell lymphoma or leukemia. In one embodiment, the tumor is a solid tumor. The solid tumor may be melanoma, colon cancer, breast cancer and/or brain tumor. In one aspect, the cancer treated is a carcinoma, sarcoma, neuroblastoma, cervical cancer, hepatocellular carcinoma, mesothelioma, glioblastoma, myeloma, lymphoma, leukemia, adenoma, adenocarcinoma, glioma, glioblastoma, retinoblastoma, astrocytoma, oligodendroglioma, meningioma, or melanoma.

The methods can be used to treat subjects such as humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), farm animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). The mammal may be of any age or at any stage of development (e.g., an adult, adolescent, child, infant or intrauterine mammal). The mammal may be male or female. In certain embodiments, the subject has or is suspected of having a neoplastic disease, neoplasia, tumor, malignancy, or cancer.

The methods disclosed herein may further comprise, consist essentially of, or consist of the steps of: administering to the subject an anti-tumor therapy other than CAR therapy.

The disclosure also relates to methods for inhibiting proliferation of a cancer cell or cancer stem cell, comprising, consisting essentially of, or consisting of: contacting the cell with an effective amount of a CAR-expressing cell, an effective amount of an antibody, an effective amount of an antigen-binding fragment, and/or an effective amount of a polypeptide disclosed herein. The CAR may be an anti-Lym-1, anti-Lym-2, or anti-CD 19-expressed CAR.

Further provided herein are methods for determining whether a subject is likely to respond or is unlikely to respond to treatment, comprising, consisting essentially of, or consisting of: contacting a sample isolated from a patient with an antibody, antigen-binding fragment, and/or polypeptide disclosed herein, and detecting the antibody-cell complex, antigen-binding fragment-cell complex, and/or polypeptide-cell complex in the sample, wherein the presence of the complex indicates that the subject is likely to respond to treatment and the absence of the complex indicates that the subject is unlikely to respond to treatment. The antibody, antigen-binding fragment, and/or polypeptide may be detectably labeled. Also further disclosed herein is a method comprising, consisting essentially of, or consisting of: administering an effective amount of an antibody or CAR disclosed herein to a subject determined to be likely to respond to treatment.

Further disclosed herein are methods directed to monitoring therapy for a subject, comprising, consisting essentially of, or consisting of: contacting a sample isolated from the subject with an antibody or antigen-binding fragment disclosed herein, and detecting the antibody-cell complex in the sample. The method can be performed before and/or after administering an effective amount of a CAR-expressing cell, an effective amount of an antibody, an effective amount of an antigen-binding fragment, and/or an effective amount of a polypeptide disclosed herein to the subject. The CAR may be an anti-Lym-1, anti-Lym-2, or anti-CD 19 expressing CAR. In one aspect, the CAR, antibody, or antigen-binding fragment thereof is detectably labeled. In another aspect, the sample comprises one or more of sputum, serum, plasma, lymph, cyst fluid, urine, feces, cerebrospinal fluid, ascites, blood, or tissue.

Also provided herein are kits for practicing the methods disclosed herein and instructions for practicing the methods disclosed herein. The kit comprises, consists essentially of, or consists of: one or more of the antibodies, CARs, antigen-binding fragments, polypeptides, isolated nucleic acids, vectors, isolated cells, and/or compositions disclosed herein and instructions for use.

Drawings

FIG. 1: the two humanized Lym-1 antibodies have different binding capacities for epitope positive cells. Will be 5X 10⁵Individual Raji cells were incubated with the indicated antibodies at concentrations increasing from 0.013 to 1300 nM. After detection with (AF 488) conjugated goat anti-human IgG, Mean Fluorescence Intensity (MFI) was quantified by flow cytometry.

FIG. 2: successful production of CAR T cells. On day 10, CAR T cell preparations were labeled with biotin-anti-Lym-1 antibody and then detected with APC-streptavidin. Blank T cells were used as negative controls.

FIGS. 3A-3C: both humanized Lym-1 CAR-T based cells induced complete remission in Raji xenografts. One million Raji/Luc-eGFP cells were injected intravenously into 8-10 week old male NSG mice (day 0). Luciferase activity was measured on day 6 to assess tumor burden prior to treatment. (figure 3A) ten million blank and CAR T cells (50% CAR positive) in 100 μ Ι PBS were injected intravenously into a randomly generated group of tumor-bearing mice (n = 5) on day 7. (fig. 3B) mean bioluminescent radiation from the dorsal and ventral images was quantified and summed. (FIG. 3C) Kaplan-Meier plot shows the survival rate of mice from control and experimental groups.

FIG. 4: production of a functional humanized Lym-1 antibody. Will be 5X 10⁵Individual ARH-77 cells were incubated with the indicated antibodies at concentrations increasing from 2.218 to 266 nM. After detection with (AF 488) conjugated goat anti-human IgG, flow cytometry was performedQuantity average fluorescence intensity (MFI).

FIG. 5: successful production of huLym-1 and huLym-2 CAR-T cells. On day 10, CAR T cell preparations were labeled with biotin-protein-L and then detected with APC-streptavidin. Blank T cells were used as negative controls.

FIG. 6: huLym-2CAR showed increased cytotoxicity against epitope positive cell lines. Blank or CAR T cells were incubated with ARH-77 eGFP/Luc at an effector/target (E/T) ratio of 2 to 0.25. Cytotoxicity was calculated from luciferase activity 17 hours after co-cultivation.

FIG. 7: schematic representation of 41BB3z and DAP10-12 based CAR T cell constructs.

FIG. 8: successful production of CAR-T cells. On day 10, CAR T cell preparations were labeled with biotin-anti-Lym-1 antibody and then detected with APC-streptavidin. Blank T cells were used as negative controls.

FIGS. 9A-9B: stable gene expression and cell proliferation of huLym-1-B-DAP transduced CAR T cells. Primary human T cells were activated on day 0 with anti-CD 3/CD28 magnetic beads (Dynabeads) and transduced with each construct on day 3. 100 ten thousand T cells in each group were restimulated with anti-CD 3/CD28 on days 7 and 14. (FIG. 9A) the absolute cell numbers in the blank and CAR-T cell preparations were counted on days 4, 7, 8 and 12. (FIG. 9B) CAR expression was measured on days 7, 14, and 21.

FIG. 10: epitope-driven cytotoxicity against Raji cells. Blank or CAR T cells were incubated with Raji-eGFP/Luc at an effector/target (E/T) ratio of 2 to 0.25. Cytotoxicity was measured according to luciferase activity 17 hours after co-cultivation.

FIGS. 11A-11D: dorsal bioluminescence imaging of tumor burden and weight changes in blank and CAR treated mice. One million Raji/Luc-eGFP cells were injected intravenously into 8-10 week old male NSG mice (day 0). Luciferase activity was measured on day 7 to assess tumor burden prior to treatment. On day 8, 100 ten thousand blanks in 100 μ Ι PBS and CAR T cells were injected intravenously into a randomly generated tumor-bearing group of mice (n = 5). (fig. 11A) example bioluminescent images. (FIG. 11B) Kaplan-Meier survival images of mice. (fig. 11C) quantification and summation of mean bioluminescent radiation from dorsal and ventral images. (FIG. 11D) weight change images of treated mice over time. The body weight of each mouse on day 0 was used as a reference for calculating the percent change in body weight. The body weight of the mice was measured twice a week with a digital scale.

FIG. 12: experiments showing DAP CD19 CAR T cells for the treatment of disseminated Raji lymphoma in NSG mice. Mice were treated with 100 million CAR T cells of huLym-1 DAP, CD19 second generation, or CD19 DAP 8 days after intravenous injection of Raji. Mice were then imaged weekly by bioluminescence and the results showed that all CD19 CAR T cells with the second generation construct died, but those receiving the CD19 DAP CAR construct remained viable at 21 days, although at this dose they still had tumors.

FIGS. 13A-13C: both Lym-1 and huLym-1-B CAR, having the intracellular domain of 4-1BB3z, can eliminate Raji tumors in vivo. (FIG. 13A) schematic of in vivo study schedule. (FIG. 13B) inoculation 10 on day 0⁶Raji-eGFP/Luc, then 5X 10 at day 6⁶Bioluminescence images of NSG mice treated with individual naive T cells, Lym-1-BB3z CAR T cells, huLym-1-B-BB3z CAR T cells, or 100. mu.l PBS. Bioluminescent signals from dorsal and ventral images of each NSG mouse were added and plotted against time. (n =5 mice/group, data not shown). (FIG. 13C) Kaplan-Meier survival Curve (.)p<0.001 by log rank test).

FIGS. 14A-14F: effect of intracellular signaling domain on huLym-1-B CAR T cell expansion. (figure 14A) in vitro expansion of blank or CAR positive T cells after transduction and restimulation (summary results from five donors). (FIG. 14B) CAR expression on T cell preparations at the days indicated. T cells were reactivated with stimulators of alpha-CD 2/CD3/CD 28-based antibodies on days 7 and 14. (figure 14C) in vitro expansion of blank or CAR positive T cells after transduction and restimulation (summary results from 3 donors). (FIG. 14D) CAR expression on T cell preparations at the days indicated. T cells were reactivated with alpha-CD 2/CD3/CD28 stimulator on days 7 and 14 (representative results from 3 donors). (figure 14E) in vitro expansion of blank or CAR positive T cells after transduction and restimulation (summary results from 3 donors). (FIG. 14F) CAR expression on T cell preparations at the days indicated. T cells were reactivated with α -CD2/CD3/CD28 stimulators on days 7 and 14 (representative results from five donors).

FIGS. 15A-15B: ligand-dependent robust signaling is the major mechanism of impaired T cell proliferation of huLym-1-B-BB3z CAR. (fig. 15A) annexin v (annexin v) and dead cell cyanine (Sytox-Green) staining (representative results from two donors) of blank or CAR transduced T cells from day 9 were evaluated. (FIG. 15B) CD19-BB3zCAR T cells were pre-labeled with Cell trace far-red dye (Cell trace face-red dye) and then co-cultured overnight with blank or indicated CAR T cells at a ratio of 1: 1. CD19-BB3zCAR T cells were then stained with annexin V (annexin V) and cyanine Green (Sytox-Green) to measure potential self-killing of huLym-1-B-BB3z or huLym-1-B-DAP CAR T cells (representative results from two donors).

FIGS. 16A-16B: huLym-1-B-DAP CAR T cells showed activation-induced reduction in cell death. Blank, huLym-1-B-BB3z and huLym-1-B-DAP CAR T cells were cultured in the presence of a-CD2/3/28 stimulus, or overnight with K562 cells or Raji cells. Blank and CAR T cells were then stained for annexin V and green cyanine (dead cell dye). (FIG. 16A) representative plot from one of three donors. (FIG. 16B) the percentage of annexin V positive cells in live cells was quantified by the following formula: Q3/(Q3 + Q4) (from three donors, P <0.05 by Student's t test bar shows mean ± SD).

FIGS. 17A-17C: huLym-1-B-DAP exhibits effector function against a panel of B lymphoma and leukemia cell lines. (FIG. 17A) cytokines released from blank, huLym-1-B-DAP and huLym-1-B-BB3z when co-cultured overnight with a panel of human lymphoma B cell lines at an effector to target ratio of 1: 1. Quantification of secreted cytokines was normalized to pg/10000 cells (GM-CSF) or ng/10000 cells (IL-2 and INF- γ) (n =3 technical replicates representative results from two donors). (FIG. 17B) to measure the cytotoxicity of blank, huLym-1-B-DAP and huLym-1-B-BB3z, tumor cells were pre-labeled with Cell trace far-red dye (Cell trace face-red dye) and then co-cultured with effector cells at a ratio of 1: 1; live tumor cells were gated at each time point. (FIG. 17C) the percentage of viable tumor cells was quantified and plotted. The percentage lysis was calculated as (% tumor at 1 hour [% tumor at 48 hours)/(% tumor at 1 hour) (n =3 technical replicates and representative results from two donors are shown.

FIGS. 18A-18B: the Lym-1 epitope was not significantly down-regulated in the presence of huLym-1-B-DAP CAR T cells. Blanks T, huLym-1-B-DAP and CD19-BB3z were co-cultured with a panel of B lymphoma and leukemia cell lines at a 1:2 ratio. After overnight co-culture, the B cell lines were labeled with antibodies against CD22, Lym-1, and CD 19.

FIGS. 19A-19D: the CAR signal domain does not affect antigen regulation. (FIG. 19A) blank T, CD19-BB3z, CD19-DAP, huLym-1-B-BB3z and huLym-1-B-DAP CAR T cells were co-cultured overnight with Raji-eGFP/Luc cells at a ratio of approximately 1: 2. CD19 antigen and Lym-1 epitope expression in residual live Raji cells was then measured by flow cytometry via fluorophore-conjugated monoclonal antibodies. (FIG. 19B) scattergrams of Mean Fluorescence Intensity (MFI) of Lym-1 epitope and CD19 antigen expression in residual Raji cells. Percentage of live Raji cells (fig. 19C) and concentration (fig. 19D) after overnight co-culture (n =3 technical replicates from two donors representative results).

FIGS. 20A-20F: huLym-1-B-DAP CAR mediated tumor-free survival at lower doses. (FIG. 20A) blank and CD19-DAP T cells were co-cultured overnight with Raji-eGFP/Luc cells at a ratio of 1: 2. The monoclonal antibody CD19 antigen conjugated by the fluorophore and the Lym-1 epitope expression were then measured by flow cytometry (representative results from three replicates). (FIG. 20B) schematic representation of in vivo studies. (FIG. 20C) 10 on day 0⁶Each Raji-eGFP/Luc was inoculated, followed by a blank, huLym-1-B-DAP, CD19-BB3z or CD19-DAP at the indicated dose on day 8 Bioluminescence image of CAR T cell treated NSG mice (n =5 mice per group). (fig. 20D) Kaplan-Meier survival curve (ns ═ log rank (Mantel-Cox) test not significant). (FIG. 20E) use 10 on day 0⁶The individual Raji-eGFP/Luc were inoculated and then inoculated with 10 on day 8⁶Bioluminescence images of blank, CD19-BB3z or CD19-DAP CAR T cell treated NSG mice (n =5 mice/group). This experiment was performed side by side with the experiment in fig. 25E, using the same blank T control group. (FIG. 20F) Kaplan-Meier survival Curve (examined by Log-rank (Mantel-Cox))P＝0.002）。

FIGS. 21A-21C: huLym-1-B was selected as a candidate for CAR development. (FIG. 21A) binding ability of a panel of 12 humanized Lym-1 antibodies to Raji cells. All antibodies were expressed as human IgG1 isotype. huLym-1-60 has the same sequence as chLym-1. hu51 is an internally produced human IgG1 isotype control. Raji cells were incubated at the indicated concentrations for 30 min and then detected with AF-488 conjugated goat anti-human IgG. Mean Fluorescence Intensity (MFI) was assessed by flow cytometry to compare the differential binding of the antibodies. (FIG. 21B) the binding capacity of the internally produced huLym-1-B and chimeric Lym-1 (chLym-1) on Raji cells was evaluated by flow cytometry. The secondary antibody used here was an APC-conjugated mouse anti-human IgG monoclonal antibody. (FIG. 21C) binding of chLym-1 and huLym-1-B to a panel of chLym-1 positive and negative cell lines. For these measurements, APC-conjugated mouse anti-human IgG monoclonal antibodies were used as secondary antibodies.

FIGS. 22A-22H: lym-1 and huLym-1-B CAR T cells with the BB3z-CD3z signaling domain are cytotoxic in culture despite impaired proliferation. (FIG. 22A) schematic representation of CAR construct. The 261 tag is a 10 amino acid linear epitope derived from the schedule of human placental growth factor (fig. 22B) CAR-T production and amplification. (figure 22C) flow cytometry analysis of CAR expression on blank or transduced primary human T cells at day 7; CAR expression was measured against 261 tag by Dylight 650 conjugated antibody. (FIG. 22D) cytotoxicity of CAR T cells at the Effector-to-target (E: T) ratios shown for Lym-1 epitope negative (K562) and positive (Raji) cell lines(ii) a Day 9 CAR T cells were used to measure cytotoxicity. No restimulation was performed on day 7 of the experiment (n = 3 technical replicates representing results from 3 donors). (FIG. 22E) fold expansion of in vitro cultured blank or CAR positive T cells from day 7-14 (summary results from 6 donors. ns-not significant. by Student's T testP<0.001). (FIG. 22F) shows day 9 CD3

Detailed description of the preferred embodiments

The following embodiments specifically describe various aspects of the present disclosure.

An antibody, comprising:

a. a Heavy Chain (HC) immunoglobulin variable domain sequence comprising SEQ ID NO: 2 or 6 or an equivalent of each thereof; and/or

b. A Light Chain (LC) immunoglobulin variable domain sequence comprising SEQ ID NO: 4 or 8 or an equivalent of each thereof.

The antibody as described herein, wherein the Heavy Chain (HC) immunoglobulin variable domain sequence comprises the amino acid sequence SEQ ID NO: 2 and the Light Chain (LC) immunoglobulin variable domain sequence comprises the amino acid sequence SEQ ID NO: 4, or an equivalent of each thereof.

The antibody as described herein, wherein the Heavy Chain (HC) immunoglobulin variable domain sequence comprises the amino acid sequence SEQ ID NO: 2 and the Light Chain (LC) immunoglobulin variable domain sequence comprises the amino acid sequence SEQ ID NO: 8, or an equivalent of each thereof.

The antibody as described herein, wherein the Heavy Chain (HC) immunoglobulin variable domain sequence comprises amino acid sequence SEQ ID NO 6 and the Light Chain (LC) immunoglobulin variable domain sequence comprises amino acid sequence SEQ ID NO 4, or an equivalent of each thereof.

The antibody as described herein, wherein the Heavy Chain (HC) immunoglobulin variable domain sequence comprises amino acid sequence SEQ ID NO 6 and the Light Chain (LC) immunoglobulin variable domain sequence comprises amino acid sequence SEQ ID NO 8, or an equivalent of each thereof.

An antibody, comprising:

a. a Heavy Chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of SEQ ID NO 10 or an equivalent thereof; and/or

b. A Light Chain (LC) immunoglobulin variable domain sequence comprising the amino acid sequence of SEQ ID NO 12 or an equivalent thereof.

The antibody as described herein, wherein the antibody is an IgA, IgD, IgE, IgG, or IgM antibody.

The antibody as described herein, wherein the antibody comprises a constant region.

The antibody as described herein, wherein the constant region comprises an IgA, IgD, IgE, IgG or IgM constant region.

The antibody as described herein, wherein the constant region is an IgGl constant region or an Ig κ constant region.

An antibody that competes for binding with an antibody as described herein.

An antibody as described herein, wherein the antibody is a polyclonal, monoclonal or humanized antibody.

An antibody as described herein, wherein an equivalent comprises a polypeptide having at least 80% amino acid identity to the polypeptide, or a polypeptide encoded by a polynucleotide that hybridizes under highly stringent conditions to the complement of a polynucleotide encoding the polypeptide.

An antigen-binding fragment of an antibody as described herein.

An antigen binding fragment as described herein, wherein the antigen binding fragment is selected from the group consisting of Fab, F (ab ') 2, Fab', scFv, and Fv.

A polypeptide comprising SEQ ID NO: 2. 4, 6, 8, 10 or 12, or an equivalent of each thereof.

A Chimeric Antigen Receptor (CAR) comprising: (a) an antigen binding domain of the antibody, (b) a hinge domain, (c) a transmembrane domain, and (d) an intracellular signaling domain or a DAP domain.

A Chimeric Antigen Receptor (CAR) comprising: (a) an antigen binding domain of an antibody, (b) a hinge domain, (c) a transmembrane domain, and (d) an intracellular DAP 10 and/or DAP 12 domain. In one aspect, (d) comprises intracellular DAP 10 and DAP 12 domains. In another aspect, the antibody is selected from an anti-Lym 1, anti-Lym 2, or anti-CD 19 antibody. In another aspect, the antigen binding domain comprises a linker polypeptide, e.g., a polypeptide of sequence (GGGGS) n, wherein n is an integer from 1 to 6, between the HC variable domain and the LC variable domain of the antibody. The CAR can further comprise a linker polypeptide positioned between (a) to (d).

The CAR as described herein, further comprising a signal polypeptide at the amine terminus of the antigen binding domain of the antibody.

The CAR as described herein, further comprising one or more co-stimulatory signaling regions.

The CAR as described herein, wherein the hinge domain comprises a CD8 a or IgGl hinge domain, the transmembrane domain comprises a CD28 or CD8 a transmembrane domain, and the one or more co-stimulatory signaling regions transduce a signal selected from the group consisting of CD27, CD28, 4-IBB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD27, LIGHT, NKG2C, and B7-H3; and the intracellular signaling domain comprises a CD3 zeta signaling domain.

The CAR as described herein, further comprising a linker polypeptide located between the HC variable domain and the LC variable domain.

The CAR as described herein, wherein the DAP domain is DAP10 and/or DAP 12.

The CAR as described herein, further comprising a peptide AVPPQQWALS inserted after the HC and the LC variable domain.

The antibody as described herein or the CAR as described herein, further comprising a detectable label or a purification label.

An isolated nucleic acid sequence, wherein the nucleic acid sequence comprises a sequence selected from any of the sequences disclosed herein, or an equivalent of each thereof, and optionally is operably linked to a promoter and/or enhancer element.

An isolated nucleic acid sequence encoding an antibody or CAR as described herein.

An isolated nucleic acid sequence as described herein, further comprising a signal peptide polynucleotide sequence upstream of the antigen binding domain of the antibody.

The isolated nucleic acid sequence as described herein, wherein the isolated nucleic acid encoding the CAR further comprises a Kozak consensus polynucleotide sequence upstream of the antigen binding domain or enhancer of the antibody.

The isolated nucleic acid sequence as described herein, wherein the isolated nucleic acid encoding the CAR further comprises a polynucleotide sequence encoding a 2A self-cleaving peptide (T2A) upstream of the antigen binding domain of the antibody.

The isolated nucleic acid sequence as described herein, wherein the isolated nucleic acid encoding the CAR further comprises a polynucleotide sequence encoding antibiotic resistance.

The isolated nucleic acid sequence as described herein, wherein the isolated nucleic acid encoding the CAR further comprises a switching mechanism for controlling expression and/or activation of the CAR.

A vector comprising an isolated nucleic acid sequence as described herein.

A vector as described herein, wherein the vector is a plasmid or a viral vector.

A vector as described herein, wherein the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

The vector as described herein, wherein the vector is a CRISPR vector.

An isolated cell comprising an antibody and/or CAR and/or an isolated nucleic acid and/or vector described herein. In one aspect, the isolated cell is an immune cell, e.g., a mouse.

A composition comprising a vector and one or more of an antibody and/or antigen-binding fragment, and/or a polypeptide and/or a CAR and/or an isolated nucleic acid and/or a vector and/or an isolated cell described herein.

A method of producing an antibody as described herein, comprising culturing an isolated cell as described herein, wherein the isolated cell is optionally a mammalian cell.

A method of generating an anti-Lym CAR-expressing cell comprising: introducing a nucleic acid sequence encoding an anti-Lym expressing CAR as described herein into a population of immune cells; and selecting a subpopulation of immune cells that have successfully transduced the nucleic acid sequence. In one aspect, the immune cell is a T cell or NK cell. In one aspect, the immune cell population has been modified to reduce or eliminate expression of endogenous immune cell receptors, e.g., wherein the immune cell population is modified using methods employing RNA interference or CRISPR.

A method of inhibiting tumor growth and/or treating cancer and/or preventing cancer recurrence in a subject in need thereof, comprising administering to the subject an effective amount of a CAR-expressing cell produced according to a method as disclosed herein, an effective amount of an antibody as described herein, an effective amount of an antigen-binding fragment as described herein, and/or an effective amount of one or more of a polypeptide as described herein. These methods may be combined with traditional therapies such as tumor resection or traditional chemotherapy.

A method of inhibiting tumor growth and/or treating cancer and/or preventing cancer recurrence in a subject in need thereof, comprising administering to the subject an effective amount of CAR-expressing cells produced according to a method as described herein, an effective amount of an antibody as described herein, an effective amount of an antigen-binding fragment as described herein, and/or an effective amount of one or more of a polypeptide as described herein, in combination with an anti-cancer therapeutic, a checkpoint inhibitor, a regulatory T cell (Treg), a myeloid-derived suppressor cell (MDSC), fluorouracil (5-FU), a Histone Deacetylase (HDAC) inhibitor, IL-12 therapy, CpG (TLR 9 agonist), and/or an interferon gene stimulator (ing) pathway agonist.

The method as described herein, wherein the cells expressing the CAR are autologous or allogeneic to the subject being treated, and optionally is a first line, second line, third line, fourth line, or fifth line therapy. In one aspect, wherein the tumor or cancer cell expresses or overexpresses CD 19, Lym1, and/or Lym 2. In another aspect, the cancer or tumor is selected from a carcinoma, sarcoma, or leukemia. In another aspect, the tumor or cancer is a B cell lymphoma or leukemia. In another aspect, the tumor is a solid tumor. In another aspect, the solid tumor is melanoma, colon cancer, breast cancer, and/or brain tumor. In another aspect, the subject is a human, an animal, a non-human primate, a dog, a cat, a sheep, a mouse, a horse, or a cow. In another aspect, the method further comprises administering to the subject an anti-tumor therapy other than CAR therapy.

A method of inhibiting proliferation of a cancer cell or cancer stem cell, comprising contacting the cell with an effective amount of a CAR-expressing cell produced according to the methods described herein, an effective amount of an antibody as described herein, an effective amount of an antigen-binding fragment as described herein, and/or an effective amount of a polypeptide as described herein.

A method of determining whether a subject is likely to respond or unlikely to respond to a therapy, comprising contacting a sample isolated from a patient with an antibody as described herein, an antigen-binding fragment as described herein, and/or a polypeptide as described herein, and detecting an antibody-cell complex, an antigen-binding fragment-cell complex, and/or a polypeptide-cell complex in the sample, wherein the presence of a complex indicates that the subject is likely to respond to the therapy and the absence of a complex indicates that the subject is unlikely to respond to the therapy. In one aspect, the antibody, antigen-binding fragment, and/or polypeptide is detectably labeled.

The method as described herein, further comprising administering to a subject determined to be likely to respond to treatment an effective amount of one or more of an antibody as described herein or a CAR as described herein. The therapy is a first-line, second-line, third-line, fourth-line or fifth-line therapy.

A method for monitoring therapy in a subject, comprising contacting a sample isolated from the subject with an antibody or antigen-binding fragment thereof as described herein, and detecting the antibody-cell complex in the sample. When the antibody is an anti-Lym antibody, the method is performed before and/or after administering an effective amount of an anti-Lym CAR-expressing cell produced as described herein, an effective amount of an antibody as described herein, an effective amount of an antigen binding fragment as described herein, and/or an effective amount of one or more of a polypeptide as described herein. In one aspect, wherein the antibody or antigen binding fragment thereof is detectably labeled. In another aspect, the sample comprises one or more of sputum, serum, plasma, lymph, cyst fluid, urine, feces, cerebrospinal fluid, ascites, blood, or tissue.

A kit comprising one or more of an antibody as described herein, a CAR as described herein, an antigen-binding fragment as described herein, a polypeptide as described herein, an isolated nucleic acid as described herein, a vector as described herein, an isolated cell as described herein, and/or a composition as described herein, and instructions for use. Instructions for use provide guidance for performing the methods described herein.

Equivalents of the formula

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

The present techniques illustratively described herein suitably may be practiced in the absence of any element, limitation, or limitations that is not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed broadly and not restrictively. Furthermore, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the technology claimed.

Thus, it should be understood that the materials, methods, and examples provided herein represent preferred aspects, are exemplary, and are not intended to limit the scope of the present technology.

The present technology has been described broadly and generally herein. Each of the narrower species and subgeneric groupings of the general disclosure also form part of the present technology. This includes the generic description of the technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Further, where features or aspects of the technology are described in terms of Markush groups, those skilled in the art will recognize that the technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

Other aspects are set forth in the following claims.

Sequence listing

SEQ ID NO: 1 huLym-1-A VH

GAGGTGCAGCTGGTTGAATCTGGCGGCGGACTTGTGAAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTTAGCCTGACATCTTATGGCGTGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGCTGGTGGTCATTTGGAGCGACGGCAGCACCACCTACAACAGCGCCCTGAAGTCCCGGTTCACCATCAGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGCCACTACGGCTCTACCCTGGCCTTTGCTTCTTGGGGCCAGGGCACACTGGTCACAGTTTCTAGC

2 huLym-1-A VH amino acid sequence of SEQ ID NO

EVQLVESGGGLVKPGGSLRLSCAASGFSLTSYGVHWVRQAPGKGLEWLVVIWSDGSTTYNSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHYGSTLAFASWGQGTLVTVSS

SEQ ID NO: 3 huLym-1-A VL

GATATTGTGCTGACACAGAGCCCCAGCAGCCTGTCTGCTTCTCCTGGACAGAGAGTGACCATCAGCTGCAGAGCCAGCGTGAACATCTACAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGACAGGCTCCTAAGCTGCTGGTGTACAACGCCAAGATTCTGGCCGAGGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCTCTGGCCTGCAGCCTGAGGACGAGGCCGATTACTATTGCCAGCACCACTATGGCACCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG

huLym-1-A VL amino acid SEQ ID NO

DIVLTQSPSSLSASPGQRVTISCRASVNIYSYLAWYQQKPGQAPKLLVYNAKILAEGVPDRFSGSGSGTDFTLTISGLQPEDEADYYCQHHYGTFTFGGGTKLEIK 4

SEQ ID NO: 5 huLym-1-B VH

GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTGCAGCCTGGCAGATCTCTGAGACTGACCTGTACCGCCAGCGGCTTTAGCCTGACAAGCTATGGCGTGCACTGGGTCCGACAGCCTCCAGGCAAAGGACTGGAATGGCTGGCCGTGATTTGGAGCGACGGCAGCACCACATACAACAGCGCCCTGAAGTCCCGGCTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCGCCAGACACTACGGCTCTACCCTGGCCTTTGCTTCTTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT

6 huLym-1-B VH amino acid sequence of SEQ ID NO

EVQLVESGGGLVQPGRSLRLTCTASGFSLTSYGVHWVRQPPGKGLEWLAVIWSDGSTTYNSALKSRLTISKDNSKSQVYLQMNSLRAEDTAVYYCARHYGSTLAFASWGQGTLVTVSS

SEQ ID NO: 7 huLym-1-B VL

GACATCCAAATGACCCAAAGCCCTTCCTCCCTAAGTGCGTCTGTCGGGGATCGTGTGACCATAACGTGTAGAGCTTCCGTTAATATATACAGTTATTTGGCCTGGTATCAACAAAAACCAGGTAAGGCCCCAAATCTGCTTATTTACAACGCAAAAATACTTGCTGAGGGCGTTCCATCTAGATTCAGCGGGAGTGGAAGTGGTACAGATTTTACGCTTACCATAAGTTCACTGCAACCTGAGGACTTCGCCTCTTACTACTGTCAACATCATTATGGGACGTTTACCTTTGGGCAAGGGACTAAGGTGGAGATAAAG

8 huLym-1-B VL amino acid SEQ ID NO

DIQMTQSPSSLSASVGDRVTITCRASVNIYSYLAWYQQKPGKAPNLLIYNAKILAEGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQHHYGTFTFGQGTKVEIK

SEQ ID NO: 9 huLym-2 VH

GAGGTGCAACTGGTCGAATCCGGTGGCGGCCTTATCCAACCCGGTCGGTCTCTTCGCTTGTCCTGTTCTGGTAGTGGCTTCACTTTCAGTAACTACTGGATGAACTGGGTCAGGCAGGCTCCCGGTAAGGGGTTGGAATGGGTAGGTGAAATCAGGTTCAAGTCTCATAACTATGCTACCCATTTTGCTGAAAGTGTTAAGGGACGTTTTACTATTAGCAGAGACGACTACAAGTCTGTAGTGTACCTTCAGATGAATTCACTCCGGTCCGAAGATACCGCCGTATATTACTGTACTCGGAGAATTGGTAACTCTGACTATGACTGGTGGTATTTTGACGTCTGGGGCCAAGGCACTATGGTTACCGTCAGCTCA

10 huLym-2 VH amino acid sequence of SEQ ID NO

EVQLVESGGGLIQPGRSLRLSCSGSGFTFSNYWMNWVRQAPGKGLEWVGEIRFKSHNYATHFAESVKGRFTISRDDYKSVVYLQMNSLRSEDTAVYYCTRRIGNSDYDWWYFDVWGQGTMVTVSS

SEQ ID NO: 11 huLym-2 VL

GAGATCGTGCTGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAAAGCCAGCCAGAACGTGGGCAACAACGTGGCCTGGTATCAGCAGAAACCTGGCAAGGTGCCCAAGCTGCTGATCTACAGCGCCAGCTACAGATACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACGTGGCCACCTACTACTGCCAGCAGTACAACACATACCCCTTCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG

12 huLym-2 VL amino acid SEQ ID NO

EIVLTQSPSSLSASVGDRVTITCKASQNVGNNVAWYQQKPGKVPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYNTYPFTFGQGTKVEIK

SEQ ID NO: 13 huLym-1-A CAR

ATGGCCCTGCCTGTTACGGCCCTGCTGCTCCCGCTGGCCCTTTTGTTGCATGCAGCCAGGCCGGAGGTGCAGCTGGTTGAATCTGGCGGCGGACTTGTGAAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTTAGCCTGACATCTTATGGCGTGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGCTGGTGGTCATTTGGAGCGACGGCAGCACCACCTACAACAGCGCCCTGAAGTCCCGGTTCACCATCAGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGCCACTACGGCTCTACCCTGGCCTTTGCTTCTTGGGGCCAGGGCACACTGGTCACAGTTTCTAGCGGAGGCGGAGGATCAGGTGGCGGTGGATCTGGCGGTGGTGGTTCTGATATTGTGCTGACACAGAGCCCCAGCAGCCTGTCTGCTTCTCCTGGACAGAGAGTGACCATCAGCTGCAGAGCCAGCGTGAACATCTACAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGACAGGCTCCTAAGCTGCTGGTGTACAACGCCAAGATTCTGGCCGAGGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCTCTGGCCTGCAGCCTGAGGACGAGGCCGATTACTATTGCCAGCACCACTATGGCACCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGACCACGACGCCAGCGCCTAGGCCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

14 huLym-1-A CAR amino acid SEQ ID NO

MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFSLTSYGVHWVRQAPGKGLEWLVVIWSDGSTTYNSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHYGSTLAFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPSSLSASPGQRVTISCRASVNIYSYLAWYQQKPGQAPKLLVYNAKILAEGVPDRFSGSGSGTDFTLTISGLQPEDEADYYCQHHYGTFTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO: 15 huLym-1-B CAR

ATGGCCCTGCCTGTTACGGCCCTGCTGCTCCCGCTGGCCCTTTTGTTGCATGCAGCCAGGCCGGAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTGCAGCCTGGCAGATCTCTGAGACTGACCTGTACCGCCAGCGGCTTTAGCCTGACAAGCTATGGCGTGCACTGGGTCCGACAGCCTCCAGGCAAAGGACTGGAATGGCTGGCCGTGATTTGGAGCGACGGCAGCACCACATACAACAGCGCCCTGAAGTCCCGGCTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCGCCAGACACTACGGCTCTACCCTGGCCTTTGCTTCTTGGGGCCAGGGCACACTGGTCACCGTTTCTTCTGGAGGCGGAGGATCAGGTGGCGGTGGATCTGGCGGTGGTGGTTCTGACATCCAAATGACCCAAAGCCCTTCCTCCCTAAGTGCGTCTGTCGGGGATCGTGTGACCATAACGTGTAGAGCTTCCGTTAATATATACAGTTATTTGGCCTGGTATCAACAAAAACCAGGTAAGGCCCCAAATCTGCTTATTTACAACGCAAAAATACTTGCTGAGGGCGTTCCATCTAGATTCAGCGGGAGTGGAAGTGGTACAGATTTTACGCTTACCATAAGTTCACTGCAACCTGAGGACTTCGCCTCTTACTACTGTCAACATCATTATGGGACGTTTACCTTTGGGCAAGGGACTAAGGTGGAGATAAAGACCACGACGCCAGCGCCTAGGCCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

16 huLym-1-B CAR amino acid SEQ ID NO

MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLTCTASGFSLTSYGVHWVRQPPGKGLEWLAVIWSDGSTTYNSALKSRLTISKDNSKSQVYLQMNSLRAEDTAVYYCARHYGSTLAFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASVNIYSYLAWYQQKPGKAPNLLIYNAKILAEGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQHHYGTFTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO: 17 huLym-2 CAR

ATGGCCCTGCCTGTTACGGCCCTGCTGCTCCCGCTGGCTCTTTTGTTGCATGCAGCCAGGCCGGAGGTGCAACTGGTCGAATCCGGTGGCGGCCTTATCCAACCCGGTCGGTCTCTTCGCTTGTCCTGTTCTGGTAGTGGCTTCACTTTCAGTAACTACTGGATGAACTGGGTCAGGCAGGCTCCCGGTAAGGGGTTGGAATGGGTAGGTGAAATCAGGTTCAAGTCTCATAACTATGCTACCCATTTTGCTGAAAGTGTTAAGGGACGTTTTACTATTAGCAGAGACGACTACAAGTCTGTAGTGTACCTTCAGATGAATTCACTCCGGTCCGAAGATACCGCCGTATATTACTGTACTCGGAGAATTGGTAACTCTGACTATGACTGGTGGTATTTTGACGTCTGGGGCCAAGGCACTATGGTTACCGTCAGCTCAGGAGGCGGAGGTTCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCTGAGATCGTGCTGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAAAGCCAGCCAGAACGTGGGCAACAACGTGGCCTGGTATCAGCAGAAACCTGGCAAGGTGCCCAAGCTGCTGATCTACAGCGCCAGCTACAGATACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACGTGGCCACCTACTACTGCCAGCAGTACAACACATACCCCTTCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAGGCCGTTCCTCCACAGCAGTGGGCCCTGTCTACCACGACGCCAGCGCCTAGGCCACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

18 huLym-2 CAR amino acid SEQ ID NO

MALPVTALLLPLALLLHAARPEVQLVESGGGLIQPGRSLRLSCSGSGFTFSNYWMNWVRQAPGKGLEWVGEIRFKSHNYATHFAESVKGRFTISRDDYKSVVYLQMNSLRSEDTAVYYCTRRIGNSDYDWWYFDVWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPSSLSASVGDRVTITCKASQNVGNNVAWYQQKPGKVPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYNTYPFTFGQGTKVEIKAVPPQQWALSTTTPAPRPPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO 19 human IgD constant region, Uniprot P01880

APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

SEQ ID NO 20 human IgG1 constant region, Uniprot P01857

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO 21 human IgG2 constant region, Uniprot P01859

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO 22 human IgG3 constant region, Uniprot P01860

ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

SEQ ID NO 23 human IgM constant region, Uniprot P01871

GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

SEQ ID NO 24 human IgG4 constant region, Uniprot P01861

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO 25 human IgA1 constant region, Uniprot P01876

ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

26 human IgA2 constant region of SEQ ID NO, Uniprot P01877

ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

SEQ ID NO 27 human Ig kappa constant region, Unit prot P01834

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

28 DAP-CAR framework DNA sequence of SEQ ID NO

CCACGACGCCAGCGCCTAGGCCTCCAACACCAGCTCCAACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCAGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTTCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCCTGTGTGCCCGGCCTAGAAGATCCCCTGCTCAGGATGGCAAGGTGTACATCAACATGCCCGGCAGAGGCTACTTCCTGGGCAGACTGGTTCCTAGAGGAAGAGGCGCTGCCGAAGCCGCCACAAGAAAGCAGAGAATCACCGAGACAGAGAGCCCCTACCAAGAGCTGCAGGGCCAGAGATCCGACGTGTACAGCGACCTGAATACCCAGCGGCCTTACTACAAGTGA

1-135: CD8a hinge

136-207 CD8a transmembrane

208-276: DAP10

277-435: DAP12

29 DAP-CAR framework amino acids

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCLCARPRRSPAQDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK*

1-45: CD8a hinge

46-69 CD8a transmembrane

70-92: DAP10

93-144: DAP12

SEQ ID NO 30 huLym-1-B-DAPCAR DNA sequence

ATGGCCCTGCCTGTTACGGCCCTGCTGCTCCCGCTGGCCCTTTTGTTGCATGCAGCCAGGCCGGAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTGCAGCCTGGCAGATCTCTGAGACTGACCTGTACCGCCAGCGGCTTTAGCCTGACAAGCTATGGCGTGCACTGGGTCCGACAGCCTCCAGGCAAAGGACTGGAATGGCTGGCCGTGATTTGGAGCGACGGCAGCACCACATACAACAGCGCCCTGAAGTCCCGGCTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCGCCAGACACTACGGCTCTACCCTGGCCTTTGCTTCTTGGGGCCAGGGCACACTGGTCACCGTTTCTTCTGGAGGCGGAGGATCAGGTGGCGGTGGATCTGGCGGTGGTGGTTCTGACATCCAAATGACCCAAAGCCCTTCCTCCCTAAGTGCGTCTGTCGGGGATCGTGTGACCATAACGTGTAGAGCTTCCGTTAATATATACAGTTATTTGGCCTGGTATCAACAAAAACCAGGTAAGGCCCCAAATCTGCTTATTTACAACGCAAAAATACTTGCTGAGGGCGTTCCATCTAGATTCAGCGGGAGTGGAAGTGGTACAGATTTTACGCTTACCATAAGTTCACTGCAACCTGAGGACTTCGCCTCTTACTACTGTCAACATCATTATGGGACGTTTACCTTTGGGCAAGGGACTAAGGTGGAGATAAAGACCACGACGCCAGCGCCTAGGCCTCCAACACCAGCTCCAACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCAGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTTCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCCTGTGTGCCCGGCCTAGAAGATCCCCTGCTCAGGATGGCAAGGTGTACATCAACATGCCCGGCAGAGGCTACTTCCTGGGCAGACTGGTTCCTAGAGGAAGAGGCGCTGCCGAAGCCGCCACAAGAAAGCAGAGAATCACCGAGACAGAGAGCCCCTACCAAGAGCTGCAGGGCCAGAGATCCGACGTGTACAGCGACCTGAATACCCAGCGGCCTTACTACAAGTGA

1-63 CD8a leader sequence

64-780: huLym-1-B ScFv

781-915: CD8a hinge

916-

988-1056: DAP10

1057-1215: DAP12

31 huLym-1-B-DAPCAR amino acid SEQ ID NO

MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLTCTASGFSLTSYGVHWVRQPPGKGLEWLAVIWSDGSTTYNSALKSRLTISKDNSKSQVYLQMNSLRAEDTAVYYCARHYGSTLAFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASVNIYSYLAWYQQKPGKAPNLLIYNAKILAEGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQHHYGTFTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCLCARPRRSPAQDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK*

1-21 CD8a leader sequence

22-260: huLym-1-B ScFv

261-

306-329 CD8a transmembrane

330-352: DAP10

353-404: DAP12

32 anti-CD 19-261 tag-BB 3zCAR-DNA sequence

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGCGGCGGAGGCTCCGGCGGCGGAGGAAGCGGCGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGCCGTGCCCCCCCAGCAGTGGGCCCTGAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

1-63 CD8a leader

64-789: FMC63 ScFv

790: 261 Label

820-954 CD8a hinge

955 ℃ C.sub.1026 CD8a transmembrane

1027-1152: 4-1BB

1153-1488: CD3z

33 anti-CD 19-261 tag-BB 3zCAR-AA

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAVPPQQWALSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

1-21 CD8a leader

22-263: FMC63-ScFv

264-

274-318 CD8a hinge

319-342 CD8a transmembrane

343-384: 4-1BB

385-495: CD3z

34 anti-CD 19-261 tag-DAPCAR DNA sequence of SEQ ID NO

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGCCGTGCCCCCCCAGCAGTGGGCCCTGAGCACCACGACGCCAGCGCCTAGGCCTCCAACACCAGCTCCAACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCAGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTTCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCCTGTGTGCCCGGCCTAGAAGATCCCCTGCTCAGGATGGCAAGGTGTACATCAACATGCCCGGCAGAGGCTACTTCCTGGGCAGACTGGTTCCTAGAGGAAGAGGCGCTGCCGAAGCCGCCACAAGAAAGCAGAGAATCACCGAGACAGAGAGCCCCTACCAAGAGCTGCAGGGCCAGAGATCCGACGTGTACAGCGACCTGAATACCCAGCGGCCTTACTACAAATGA

1-63 CD8a leader

64-789: FMC63 ScFv

790: 261 Label

820-954 CD8a hinge

955 ℃ C.sub.1026 CD8a transmembrane

1027-1095: DAP10

1096-1254: DAP12

35 anti-CD 19-261 tag-DAPCAR AA sequence

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAVPPQQWALSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCLCARPRRSPAQDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK*

1-21 CD8a leader

22-263: FMC63-ScFv

264-

274-318 CD8a hinge

319-342 CD8a transmembrane

343-365: DAP10

366-417: DAP12。