Compositions and methods for treating cancer with anti-CD22 immunotherapy
阅读说明：本技术 用于用抗cd22免疫治疗来治疗癌症的组合物和方法 (Compositions and methods for treating cancer with anti-CD22 immunotherapy ) 是由 里马斯·J·奥伦塔什 迪娜·施奈德 博罗·德罗普利奇 迪米特尔·S·迪米特罗夫 朱忠玉 于 2018-10-16 设计创作，主要内容包括：公开了包含CD22抗原结合结构域的嵌合抗原受体(CAR)。还公开了涉及CAR的核酸、重组表达载体、宿主细胞、抗原结合片段和药物组合物。还公开了在对象中治疗或预防癌症的方法,以及制备CAR T细胞的方法。(Chimeric Antigen Receptors (CARs) comprising a CD22 antigen binding domain are disclosed. Also disclosed are nucleic acids, recombinant expression vectors, host cells, antigen-binding fragments, and pharmaceutical compositions that relate to the CARs. Also disclosed are methods of treating or preventing cancer in a subject, and methods of making CAR T cells.)
1. an isolated nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR) comprising at least one extracellular antigen-binding domain comprising a CD22 antigen-binding domain encoded by a nucleotide sequence comprising SEQ ID No.1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, or 171, at least one transmembrane domain, and at least one intracellular signaling domain.
2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD22 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to CD 22.
3. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD22 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD 22.
4. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD22 antigen binding domain, the at least one intracellular signaling domain, or both, are linked to the transmembrane domain by a linker or spacer domain.
5. The isolated nucleic acid molecule of claim 4, wherein the encoded linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is linked to the transmembrane domain.
6. The isolated nucleic acid molecule of claim 1, wherein the encoded extracellular CD22 antigen-binding domain is preceded by a leader nucleotide sequence encoding a leader peptide.
7. The isolated nucleic acid molecule of claim 6, wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO: 190 encoding the sequence of SEQ ID NO: 191, or a leader amino acid sequence.
8. The isolated nucleic acid molecule of claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: an α, β, or zeta chain of a T cell receptor, CD8, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.
9. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the extracellular CD22 antigen binding domain comprises a nucleic acid sequence comprising SEQ ID No.1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, or 171 or a sequence 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
10. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain further comprises a CD3 ζ intracellular domain.
11. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof.
12. The isolated nucleic acid molecule of claim 11, wherein the encoded at least one co-stimulatory domain comprises a functional signaling domain that: OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof.
13. A Chimeric Antigen Receptor (CAR) encoded by the isolated nucleic acid molecule of claim 1.
14. The CAR of claim 13, comprising at least one extracellular antigen-binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, or a pharmaceutically acceptable salt thereof.
15. The CAR of claim 14, wherein the CD22 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to CD 22.
16. The CAR of claim 14, wherein the CD22 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD 22.
17. The CAR of claim 14, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: an α, β, or zeta chain of a T cell receptor, CD8, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.
18. The CAR of claim 17, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence substantially identical to SEQ ID NO: 182 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
19. The CAR of claim 14, wherein the at least one extracellular antigen-binding domain and the at least one intracellular signaling domain, or both, are linked to the transmembrane domain by a linker or spacer domain, the extracellular antigen-binding domain comprising a CD22 antigen-binding domain comprising the amino acid sequence of SEQ ID No.2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172.
20. The CAR of claim 19, wherein the linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is linked to a transmembrane domain.
21. The CAR of claim 14, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.
22. The CAR of claim 21, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of: OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.
23. A vector comprising the nucleic acid molecule of claim 1.
24. The vector of claim 23, wherein the vector is selected from the group consisting of: a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector, or a retroviral vector, or a combination thereof.
25. The vector of claim 23, further comprising a promoter.
26. The vector of claim 25, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue specific promoter, a suicide type promoter, or any combination thereof.
27. A cell comprising the vector of claim 23.
28. The cell of claim 27, wherein the cell is a T cell.
29. The cell of claim 28, wherein the T cell is a CD8+ T cell.
30. The cell of claim 27, wherein the cell is a human cell.
31. A method of making a cell comprising transducing a T cell with the vector of claim 23.
32. A method of producing a population of RNA engineered cells comprising introducing in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises the nucleic acid molecule of claim 1.
33. A method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of the cell of claim 27.
34. A method of treating or preventing cancer in a mammal comprising administering to the mammal the CAR of claim 13 in an amount effective to treat or prevent cancer in the mammal.
35. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises at least one extracellular antigen-binding domain comprising a CD22 antigen-binding domain comprising the amino acid sequence of SEQ ID No.2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, at least one linker domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a human having cancer.
36. The pharmaceutical composition of claim 35, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: an α, β, or zeta chain of a T cell receptor, CD8, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.
37. The pharmaceutical composition of claim 35, wherein the T cell is a T cell of a human having a hematologic cancer.
38. The pharmaceutical composition of claim 37, wherein the hematologic cancer is leukemia or lymphoma.
39. The pharmaceutical composition of claim 38, wherein the leukemia is Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), or Chronic Myelogenous Leukemia (CML).
40. The pharmaceutical composition of claim 38, wherein the lymphoma is mantle cell lymphoma, non-hodgkin's lymphoma (NHL), or hodgkin's lymphoma.
41. The pharmaceutical composition of claim 37, wherein the hematologic cancer is multiple myeloma.
42. The pharmaceutical composition of claim 35, wherein the human cancer comprises adult cancers including cancers of the oral and pharyngeal cavity (tongue, mouth, pharynx, head and neck), digestive system (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gall bladder, pancreas), respiratory system (larynx, lung and bronchi), bone and joint, soft tissue, skin (melanoma, basal and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or any combination thereof.
43. A method of treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain comprising a CD22 antigen binding domain comprising the amino acid sequence of SEQ ID No.2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, at least one transmembrane domain, at least one intracellular signaling domain, wherein the T cells are T cells of a subject having cancer.
44. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain comprising a CD22 antigen binding domain comprising the amino acid sequence of SEQ id No.2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, wherein the T cells are T cells of a subject having cancer.
45. The method of claim 43 or 44, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: an α, β, or zeta chain of a T cell receptor, CD8, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.
46. A method for producing a cell expressing a Chimeric Antigen Receptor (CAR), the method comprising introducing into the cell the isolated nucleic acid of claim 1.
47. The method for producing a Chimeric Antigen Receptor (CAR) -expressing cell according to claim 46, wherein the cell is a T cell or a population of cells comprising a T cell.
Technical Field
The present application relates to the field of cancer, in particular to CD22 antigen binding domains and Chimeric Antigen Receptors (CARs) comprising such CD22 antigen binding domains and methods of use thereof.
Background
Cancer is one of the most fatal threats to human health. In the united states alone, cancer affects nearly 130 million new patients each year, and it is the second cause of death following cardiovascular disease, causing approximately one-fourth of deaths. Solid tumors are the cause of most of these deaths. Despite significant advances in the medical treatment of certain cancers, the overall 5-year survival rate of all cancers has only improved by about 10% over the last 20 years. Cancer or malignant tumors metastasize and grow rapidly in an uncontrolled manner, making treatment extremely difficult.
Current standard of care for B-lineage leukemia may consist of remission induction therapy by high dose chemotherapy or radiation followed by consolidation and may be characterized by stem cell transplantation and additional chemotherapeutic procedures as needed (see world wide web cancer. The high toxicity associated with these treatments and the risk of complications such as relapse, secondary malignancy or Graft Versus Host Disease (GVHD) have prompted the search for better treatment options. CD22, also known as SIGLEC-2 (sialic acid binding immunoglobulin-like lectin 2), is a 95kDa transmembrane surface glycoprotein and comprises 6 Ig-like C2-type domains and 1 Ig-like V-type domain (uniprot. org/uniprot/P20273# structure, visited 12 months 7/2017). During B cell ontogeny, CD22 is expressed on the B cell surface starting from the pre-B cell stage, persists on mature B cells, and is lost on plasma cells (Nitschke L, 2009, immunologicals reviews, 230: 128-. CD22 contains an intracellular ITIM (immunoreceptor tyrosine-based inhibitory motif) domain that is used to down-regulate cellular activation following engagement of a B cell receptor against an antigen (engagment). Antibody binding of CD22 induces co-localization with SHP-1 and intracellular phosphatases that are also used to down-regulate phosphorylation-based signal transduction (LumbS, Fleishcer SJ, Wiedemann A, Daridon C, Maloney A, Shock A, Dorner T, 2016, journal of Cell Communication and Signaling, 10: 143-151). A key point associated with the treatment of B cell malignancies is that CD22 is expressed in a strictly regulated manner on normal B cells but not on hematopoietic stem cells or mature plasma cells, making CD22 a suitable target antigen for B cell leukemia. Expression of CD22 on both adult and pediatric (pre-B-ALL) B cell malignancies has led to the use of this target for antibody-based and chimeric antigen receptor-T cell-based therapy (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerlad DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ, 2013, Blood, 121: 1165-.
A number of new methods for treating B cell leukemias and lymphomas have been developed, including anti-CD22 antibodies linked to bacterial toxins or chemotherapeutic agents (Wayne AS, FitzGerald DJ, Kreitman RJ, Pastan I, 2014, Immunotoxins for leukamia, Blood, 123: 2470-. Olympuzumab (Inotuzumab zogamicin) (CMC-544, a humanized form of the murine monoclonal antibody G5/44) is an antibody drug conjugate, and it is currently being evaluated in clinical trials, either as a single agent or in combination with chemotherapy (NCT01664910, sponsor: M.D. Anderson Cancer Center) (DiJoseph JF, et al., 2004, Blood, 103: 1807-1814). As a single agent, the outcome outperformed that observed under standard treatment conditions, but significant hepatotoxicity was noted (Kantariian H, et al, 2016, Inotuzumab yeast strain for oral viral hepatitis leukemia (ALL), New England Journal of medicine, 375: 740-. The unmodified CD22 therapeutic antibody Epaucuzumab (Epratuzumab) is also being tested in combination with chemotherapy (NCT01219816, sponsor: Nantes University Hospital). Epratuzumab is a chimeric protein consisting of murine CDRs grafted onto a human antibody framework. Although effective in some leukemias, mosetumomabpasaudotox is not in widespread clinical development due to both the immunogenicity of the bacterial toxin fused to the antibody and the modest or comparable level of activity with other agents (see NCT01829711, sponsor: MedImmune, LLC). Many of the CD22 binding moieties used to date in CAR constructs have utilized domains derived from these murine antibodies and have not been able to effectively activate T cells targeting this CD22 domain (e.g., HA22 anti-CD22 binding agents used as the basis for Moxetemoma pasudotox, see James SE, Greenberg PD, Jensen MC, Lin Y, Wang J, Till BG, Raubitschek AA, Forman SJ, Press OW, 2008, journal of Immunology 180: 7028-. Clinical trials are currently being conducted at the National Institutes of Health (NIH) on an Anti-CD22 binding agent that is effective as an Anti-CD22 CAR, but the results have not been published (government identifiers for clinical trials: NCT02315612, Anti-CD22 ChimericReceptor T Cells in Pediatric and Young additives with a Current or RefractoryCD22-expressing B Cells Malignances, sponsor: NCI). The binding agent is based on the fully human M971 antibody developed in the laboratory of Dr Dimitrov, a Dimiter of the present inventors (Xiao X, Ho M, Zhu Z, PastanI, Dimitrov D, 2009, Identification and characterization of heavy human anti-CD22 monoclonal antibodies, MABS, 1: 297-. In work supervised by Dr Rimas Orentas, another inventor of the present application, the m971 domain proved to be effective as CAR (Haso W, et al, 2013, Anti-CD22-CARs targeting B-cell precorsor ALL, Blood, 121: 1165-.
Chimeric Antigen Receptors (CARs) are hybrid molecules comprising three basic units: (1) an extracellular antigen binding motif, (2) a ligation/transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, OrestasRJ. Lessons left from a high-active-CD 22-specific CAR. Oncoimmunogenogy.2013; 2 (4): e 23621). The antigen binding motif of a CAR generally mimics the smallest binding domain of a single chain variable Fragment (scFv), immunoglobulin (Ig) molecule. Alternative antigen binding motifs, such as receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressed IL-13 receptor), whole immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D) have also been engineered into CARs. Alternative cellular targets for CAR expression (e.g., NK or γ -T cells) are also under development (Brown CE et al Clincancer Res.2012; 18 (8): 2199-209; Lehner M et al PLoS one.2012; 7 (2): e 31210). There remains significant work to be done in defining the most active T cell population for transduction with CAR vectors, determining the optimal culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.
The linking motif of the CAR can be a relatively stable domain (e.g., the constant domain of an IgG) or a flexible linker designed to be extended. Structural motifs such as those derived from IgG constant domains can be used to extend scFv binding domains away from the T cell plasma membrane surface. This may be important for some tumor targets where the binding domain is in particular close to the surface membrane of the tumor cell (e.g. for disialoganglioside GD 2; Orentas et al, unpublished observations). To date, the signaling motif for CARs generally comprises the CD 3-zeta chain, as this core motif is a key signal for T cell activation. The first two-generation CARs reported were characterized by a CD28 signaling domain and a CD28 transmembrane sequence. This motif is also used for third generation CARs that contain the CD137(4-1BB) signaling motif (Zhao Y et al J immunol. 2009; 183 (9): 5563-74). With the new technology: activation of T cell appearance with beads linked to anti-CD 3 and anti-CD 28 antibodies the presence of the typical "signal 2" from CD28 no longer needs to be encoded by the CAR itself. By using bead activation, it was found that the tertiary vector did not outperform the secondary vector in vitro assays and that the tertiary vector did not provide significant benefit over the secondary vector in a mouse model of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, OrentasRJ.Anti-CD22-CARs targeting B cell JNL precursor ALL, blood.2013; 121: 1165-74; Kondechrer fer et al. blood.2012; 119 (12): 2709-20). In addition to CD137, other tumor necrosis factor receptor superfamily members (e.g., OX40) are also capable of providing important sustained signals in CAR-transduced T cells (Yvon Eet al. Clin Cancer Res.2009; 15 (18): 5852-60). Also important are culture conditions for culturing CAR T cell populations, e.g., the inclusion of cytokines IL-2, IL-7 and/or IL-15 (Kaiser AD et al cancer GeneTher.2015; 22 (2): 72-78).
Current challenges in broader and effective adaptation of CAR therapies for cancer are associated with the lack of powerful targets. Establishing binding agents to cell surface antigens is readily achievable today, but finding cell surface antigens that are specific for tumors while sparing normal tissues remains a formidable challenge. One potential way to confer greater target cell specificity to CAR-expressing T cells is to use a combinatorial CAR approach. In one system, the CD 3-zeta and CD28 signaling units are isolated in two different CAR constructs expressed in the same cell; in another system, both CARs are expressed in the same T cell, but one has lower affinity and therefore alternative CARs are required to be conjugated first to make the other fully active (Lanitis E et al cancer Immunol res.2013; 1 (1): 43-53; Kloss CC et al nat biotechnol.2013; 31 (1): 71-5). Another challenge in generating single scFv-based CARs as immunotherapeutics is tumor cell heterogeneity. At least one team has developed a CAR strategy for glioblastoma in which a population of effector cells simultaneously targets multiple antigens (HER2, IL-13Ra, EphA2), with the desire to avoid growth of target antigen-negative populations (Hegde Metal. mol ther.2013; 21 (11): 2087-.
T cell-based immunotherapy has become a new frontier in synthetic biology; a variety of promoters and gene products are envisioned to direct these highly potent cells to the tumor microenvironment, where T cells can escape negative regulatory signals and can mediate efficient tumor killing. Elimination of unwanted T cells by drug-induced dimerization of inducible caspase 9 constructs with chemically based dimerizers (e.g., AP1903) suggests a way in which a powerful switch that can control T cell populations can be pharmacologically turned on (Di Stasi a et al.n Engl J med.2011; 365 (18): 1673-83). The generation of effector T cell populations that are immune to the negative regulatory effects of transforming growth factor beta through the expression of decoy receptors further indicates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al. jimmunether.2008; 31 (5): 500-5). Thus, while CARs have been shown to trigger T cell activation in a manner similar to endogenous T cell receptors, the major hurdles to clinical application of this technology have so far been limited by the in vivo expansion of CAR + T cells, rapid disappearance of cells after infusion, and disappointing clinical activity. This can be partly due to the murine origin of some of the CAR sequences employed, a hurdle that is directly addressed by our invention disclosed herein.
Accordingly, there is an urgent and long-felt need in the art to find new compositions and methods for treating B-ALL, DLBCL, FL, and other CD22 expressing B cell malignancies using methods that can exhibit specific and effective anti-tumor effects without the aforementioned drawbacks.
The present invention addresses these needs by providing CAR compositions and therapeutic methods that can be used to treat cancer and other diseases and/or disorders. In particular, the invention as disclosed and described herein provides such CARs: which are useful for treating a disease, disorder or condition associated with deregulated CD22 expression, and which CAR comprises a CD22 antigen binding domain that exhibits high surface expression on transduced T cells, exhibits high cytolysis of cells expressing CD22, and wherein the transduced T cells exhibit in vivo expansion and persistence.
Summary of The Invention
Provided herein are novel anti-CD22 antibodies or antigen-binding domains thereof and Chimeric Antigen Receptors (CARs) comprising such CD22 antigen-binding domains, as well as host cells (e.g., T cells) expressing the receptors, and nucleic acid molecules encoding the receptors. CARs exhibit high surface expression on transduced T cells, have high cytolysis, and have transduced T cell expansion and persistence in vivo. Also provided are methods of using the disclosed CARs, host cells, and nucleic acid molecules, e.g., to treat cancer in a subject.
Accordingly, in one aspect, there is provided an isolated polynucleotide encoding a human anti-CD22 antibody or fragment thereof, comprising a sequence selected from the group consisting of SEQ ID NOs: 1. 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161 and 171.
In one embodimentProvided are isolated polynucleotides encoding a fully human anti-CD22 antibody or fragment thereof, wherein said antibody or fragment thereof comprises a sequence selected from the group consisting of Fab fragments, F (ab')2Fragments, Fv fragments and fragments of single-chain Fv (ScFv).
In one embodiment, an isolated polynucleotide is provided encoding a fully human anti-CD22 antibody or fragment thereof, wherein the antibody or fragment thereof comprises a sequence selected from the group consisting of SEQ ID NOs: 2. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, and 172.
In one aspect, there is provided an isolated nucleic acid molecule encoding a CAR comprising, from N-terminus to C-terminus, at least one CD22 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, the at least one CD22 antigen binding domain consisting of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1. 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161 and 171.
In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD22 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD 22.
In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD22 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD 22.
In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular CD22 antigen binding domain further comprises at least one lipocalin-based antigen binding antigen (anti-transportin) that binds to CD 22.
In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22 antigen binding domain is linked to a transmembrane domain by a linker domain.
In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CD22 extracellular antigen-binding domain is preceded by a sequence encoding a leader peptide or a signal peptide.
In another embodiment, an isolated nucleic acid molecule is provided that encodes a CAR comprising at least one CD22 antigen binding domain encoded by a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1. 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161 and 171, and wherein the CAR additionally encodes an extracellular antigen-binding domain that targets an antigen including, but not limited to: CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123(IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.
In certain embodiments, isolated nucleic acid molecules are provided that encode a CAR, wherein the additional encoded extracellular antigen-binding domain comprises an anti-CD 19 ScFv antigen-binding domain, an anti-CD 20 ScFv antigen-binding domain, an anti-ROR 1 ScFv antigen-binding domain, an anti-mesothelin ScFv antigen-binding domain, an anti-CD 33 ScFv antigen-binding domain, an anti-CD 38ScFv antigen-binding domain, an anti-CD 123(IL3RA) ScFv antigen-binding domain, an anti-CD 138 ScFv antigen-binding domain, an anti-BCMA (CD269) ScFv antigen-binding domain, an anti-GPC 2ScFv antigen-binding domain, an anti-GPC 3 ScFv antigen-binding domain, an anti-FGFR 4 ScFv antigen-binding domain, an anti-lptsr ScFv antigen-binding domain, an anti-c-ScFv antigen-binding domain, an anti-PMSA ScFv antigen-binding domain, an anti-glycolipid F77 ScFv antigen-binding domain, an anti-EGFRvIII, an anti-ScFv antigen-binding domain, an anti-ScFv antigen-binding, An anti-NY-ESO-1 TCR ScFv antigen binding domain, an anti-MAGE a3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.
In one aspect, the CARs provided herein further comprise a linker or spacer domain.
In one embodiment, an isolated nucleic acid molecule is provided that encodes a CAR, wherein the extracellular CD22 antigen binding domain, the intracellular signaling domain, or both are linked to the transmembrane domain by a linker or spacer domain.
In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 or CD28 and is linked to a transmembrane domain.
In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: an α, β, or zeta chain of a T cell receptor, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.
In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 ζ intracellular domain.
In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.
In other embodiments, isolated nucleic acid molecules are provided that encode a CAR, wherein the encoded at least one co-stimulatory domain comprises a functional signaling domain that: OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.
In one embodiment, an isolated nucleic acid molecule is provided that encodes a CAR further comprising a leader sequence or a signal peptide, wherein the leader or signal peptide nucleotide sequence comprises SEQ ID NO: 190.
In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises SEQ ID NO: 191, or a pharmaceutically acceptable salt thereof.
In one aspect, provided herein are CARs comprising, from N-terminus to C-terminus, at least one CD22 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.
In one embodiment, a CAR is provided, wherein the extracellular CD22 antigen-binding domain comprises at least one single chain variable fragment of an antibody that binds an antigen, or at least one heavy chain variable region of an antibody that binds an antigen, or a combination thereof.
In another embodiment, a CAR is provided, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: an α, β, or zeta chain of a T cell receptor, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.
In some embodiments, a CAR is provided, wherein the CAR further encodes an extracellular antigen-binding domain comprising: CD19, CD20, ROR1, mesothelin, CD33, CD38, CD123(IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, TSLPR, c-Met, PSMA, glycolipids F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or an amino acid sequence 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or any combination thereof.
In one embodiment, a CAR is provided, wherein the extracellular antigen-binding domain comprises an anti-CD 19 ScFv antigen-binding domain, an anti-CD 20 ScFv antigen-binding domain, an anti-ROR 1 ScFv antigen-binding domain, an anti-mesothelin ScFv antigen-binding domain, an anti-CD 33 ScFv antigen-binding domain, an anti-CD 38ScFv antigen-binding domain, an anti-CD 123(IL3RA) ScFv antigen-binding domain, an anti-CD 138 ScFv antigen-binding domain, an anti-BCMA (CD269) ScFv antigen-binding domain, an anti-GPC 2ScFv antigen-binding domain, an anti-GPC 3 ScFv antigen-binding domain, an anti-FGFR 4 ScFv antigen-binding domain, an anti-slpr ScFv antigen-binding domain, an anti-c-Met ScFv antigen-binding domain, an anti-PMSA ScFv antigen-binding domain, an anti-glycolipid F77 ScFv antigen-binding domain, an anti-EGFRvIII antigen-binding domain, an anti-ScFv antigen-binding domain, an anti-GD 2ScFv antigen-binding domain, An anti-NY-ESO-1 TCR ScFv antigen binding domain, an anti-MAGE a3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.
In another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.
In another embodiment, there is provided a CAR, wherein at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of: OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.
In one embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 3(LTG 2202LP-16P-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (fig. 2A)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 4 (LTG2202 LP-16P-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (fig. 2A)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 13(LTG 2246 LP-24P-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (fig. 2B)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 14 (LTG2246 LP-24P-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (fig. 2B)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 23 (LTG2247 LP-25P-CD 8TM-41BB-CD3 ζ CAR nucleotide sequence (fig. 2C)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 24 (LTG2247 LP-25P-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (fig. 2C)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 33 (LTG2248 LP-11S-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (fig. 2D)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 34 (LTG2248 LP-11S-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (FIG. 2D)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 43 (LTG2249 LP-12S-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2E)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 28 (LTG2208 LP-12S-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (FIG. 2E)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 53 (LTG2203 LP-16P3-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2F)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 54 (LTG2203 LP-16P3-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (fig. 2F)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 63 (LTG2204 LP-16P16-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2G)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 34 (LTG2204 LP-16P16-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (FIG. 2G)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 73 (LTG2205 LP-16P20-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2H)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 74 (LTG2205 LP-16P20-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (FIG. 2H)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 83 (LTG2206 LP-16P2-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2I)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 84 (LTG2206 LP-16P2-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (FIG. 2I)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 93 (LTG2207 LP-16P6-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2J)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 94 (LTG2205 LP-16P20-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (FIG. 2J)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 103 (LTG2208 LP-16P10-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (fig. 2K)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 104 (LTG2208 LP-16P10-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (fig. 2K)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 113 (LTG2209 LP-16P17-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (fig. 2L)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 114 (LTG2209 LP-16P17-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (fig. 2L)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 123 (LTG2210 LP-16P20v2-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2M)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 124 (LTG2210 LP-16P20v2-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (FIG. 2M)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 133 (LTG2216 LP-16P1-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2N)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 134 (LTG2216 LP-16P1-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (FIG. 2H)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 143 (LTG2217 LP-16P3v2-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2O)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 144 (LTG2217 LP-16P3v2-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (fig. 2O)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 153 (LTG2218 LP-16P8-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2P)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 154 (LTG2218 LP-16P8-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (FIG. 2P)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 163 (LTG2219 LP-16P13-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2Q)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 164 (LTG2219 LP-16P13-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (FIG. 2Q)).
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 173 (LTG2220 LP-16P15-CD 8TM-41BB-CD3 ζ CAR nucleic acid sequence (FIG. 2R)). In one embodiment, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO: 174 (LTG2220 LP-16P15-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (fig. 2R)).
In one aspect, the CARs disclosed herein are modified to express or comprise a detectable marker for use in diagnosing, monitoring and/or predicting treatment outcome (e.g., progression-free survival of a cancer patient) or for monitoring the progression of such treatment.
In one embodiment, the nucleic acid molecule encoding the disclosed CAR can be contained in a vector, e.g., a viral vector. The vector is a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector, or a retroviral vector, or a combination thereof.
In certain embodiments, the vector further comprises a promoter, wherein the promoter is an inducible promoter, a tissue specific promoter, a constitutive promoter, a suicide promoter (suicide promoter), or any combination thereof.
In another embodiment, the CAR-expressing vector may also be modified to include one or more manipulating elements that control CAR T cell expression or eliminate CAR-T cells via a suicide switch. Suicide switches may include, for example, drugs that induce apoptosis-inducing signaling cascades or induce cell death. In a preferred embodiment, the vector expressing the CAR may also be modified to express an enzyme, such as Thymidine Kinase (TK) or Cytosine Deaminase (CD).
In another aspect, host cells comprising a nucleic acid molecule encoding a CAR are also provided. In some embodiments, the host cell is a T cell, e.g., a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8+ T cell.
In another aspect, a pharmaceutical composition is provided comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises at least one extracellular antigen-binding domain comprising a CD22 antigen-binding domain comprising the amino acid sequence of SEQ ID No.2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the T cells are T cells of a human having cancer. The cancer includes inter alia hematological cancers (hematological cancers), such as leukemia (e.g., (CLL, ALL, AML or CML, lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, NHL) or Hodgkin's lymphoma), or multiple myeloma, or a combination thereof.
In one embodiment, a pharmaceutical composition is provided, wherein the at least one transmembrane domain of the CAR comprises a transmembrane domain of a protein selected from the group consisting of: an α, β, or zeta chain of a T cell receptor, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.
In another embodiment, a pharmaceutical composition is provided, wherein the human cancer comprises adult cancer (adultcrarcinoma), comprising: oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), digestive cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gall bladder, pancreas), respiratory cancers (larynx, lung and bronchi), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, reproductive system (cervix, corpus uteri, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or any combination thereof.
In another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of a population of human T cells of a human having a cancer, wherein the cancer is a refractory cancer that is not responsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer (myelodysplastic cancer), myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, Minimal Residual Disease (MRD) among: ALL, AML, adult B cell malignancies (including CLL, CML, NHL), pediatric B cell malignancies (including B lineage ALL), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematologic and solid tumors, or any combination thereof.
In another aspect, a method of making a T cell comprising a CAR (hereinafter "CAR-T cell") is provided. The method comprises transducing a T cell with a vector or nucleic acid molecule encoding the disclosed CAR that specifically binds CD22, thereby making a CAR-T cell.
In another aspect, there is provided a method of producing a population of RNA engineered cells comprising introducing an in vitro transcribed RNA or a synthetic RNA of a nucleic acid molecule encoding the disclosed CAR into a cell of a subject, thereby producing a CAR cell.
In another aspect, there is provided a method for diagnosing a disease, disorder or condition associated with CD22 expression on a cell, comprising: a) contacting the cell with a human anti-CD 19 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172; and b) detecting the presence of CD22, wherein the presence of CD19 diagnoses a disease, disorder or condition associated with CD22 expression.
In one embodiment, the disease, disorder or condition associated with CD22 expression is a cancer, including hematopoietic cancers, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, Minimal Residual Disease (MRD) among: ALL, AML, adult B cell malignancies (including CLL, CML, NHL), pediatric B cell malignancies (including B lineage ALL), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematologic and solid tumors, or any combination thereof.
In another embodiment, there is provided a method of diagnosing, prognosing or determining the risk of a CD 19-associated disease in a mammal, comprising detecting the expression of CD22 in a sample derived from the mammal, comprising: a) contacting the sample with a human anti-CD22 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172; and b) detecting the presence of CD22, wherein the presence of CD22 diagnoses a CD 22-associated disease in the mammal.
In another embodiment, there is provided a method of inhibiting CD 22-dependent T cell inhibition comprising contacting a cell with a human anti-CD22 antibody or fragment thereof, wherein the antibody or fragment thereof comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 2. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172. In one embodiment, the cell is selected from the group consisting of a tumor cell expressing CD22, a tumor-associated macrophage, and any combination thereof.
In another embodiment, a method of blocking T cell inhibition mediated by a cell expressing CD22 and altering the tumor microenvironment to inhibit tumor growth in a mammal is provided comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD22 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, and 172. In one embodiment, the cell is selected from the group consisting of a tumor cell expressing CD19, a tumor-associated macrophage, and any combination thereof.
In another embodiment, a method of inhibiting, suppressing or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD22 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, and 172. In one embodiment, the antibody or fragment thereof inhibits the interaction between a first cell and a T cell, wherein the first cell is selected from the group consisting of a tumor cell expressing CD22, a tumor-associated macrophage, and any combination thereof.
In another aspect, there is provided a method for inducing anti-tumor immunity in a mammal comprising administering to the mammal a therapeutically effective amount of a T cell transduced with a vector or nucleic acid molecule encoding a disclosed CAR.
In another embodiment, a method of treating or preventing cancer in a mammal is provided comprising administering to the mammal one or more disclosed CARs in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to a subject a therapeutically effective amount of a host cell expressing a disclosed CAR that specifically binds CD22 and/or one or more of the foregoing antigens under conditions sufficient to form an immune complex on the CAR with CD22 and/or the extracellular domain of one or more of the foregoing antigens in the subject.
In another embodiment, there is provided a method for treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises at least one extracellular CD22 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, the at least one extracellular CD22 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 2. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, or any combination thereof, and wherein the T cell is a T cell of a subject having cancer.
In another embodiment, there is provided a method for treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises at least one CD22 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, the at least one CD22 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 2. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, or any combination thereof, wherein the T cell is a T cell of a subject having cancer. In some embodiments of the foregoing methods, the at least one transmembrane domain comprises the transmembrane domains: an α, β, or zeta chain of a T cell receptor, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof.
In another embodiment, a method for generating a persisting population of genetically engineered T cells in a human diagnosed with cancer is provided. In one embodiment, the method comprises administering to a human a T cell genetically engineered to express a CAR, wherein the CAR comprises at least one CD22 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, the at least one CD22 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 2. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, or any combination thereof, wherein the population of persisting genetically engineered T cells or progeny of the T cells persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.
In one embodiment, the progeny T cells in a human comprise memory T cells. In another embodiment, the T cell is an autologous T cell.
In all aspects and embodiments of the methods described herein, any one of the aforementioned cancers, diseases, disorders or conditions associated with elevated expression of a tumor antigen can be treated or prevented or ameliorated using one or more CARs disclosed herein.
In another aspect, there is provided a kit (kit) for the preparation of a CAR T cell as described above or for the prevention, treatment or amelioration of any one of the cancers, diseases, disorders or conditions associated with elevated expression of a tumor antigen in a subject as described above, comprising a container comprising any one of the nucleic acid molecules, vectors, host cells or compositions disclosed above, or any combination thereof, and instructions for the use of the kit.
It is understood that CARs, host cells, nucleic acids, and methods are also useful beyond the particular aspects and embodiments described in detail herein. The foregoing features and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
Brief Description of Drawings
Figure 1 depicts a schematic diagram of the general domain structure of a CAR with a novel extracellular CD22 antigen binding domain sequence. The CAR consists of an extracellular CD22 binding ScFv domain, a CD8 spacer and transmembrane domain, an intracellular signaling CD137 costimulatory domain, and a CD3 zeta signaling domain.
Figures 2A to 2R depict several CARs comprising a novel extracellular CD22 antigen-binding domain sequence. The general scheme for CAR includes a signal peptide from N-terminus to C-terminus, an anti-CD22 binding agent heavy chain variable fragment or a linked single chain variable fragment (ScFv), an extracellular linker, a transmembrane, 4-1BB, CD3 ζ.
FIG. 2A depicts a lentiviral vector expressing a CAR comprising the LTG 220216P CD22ScFv-CD8TM-41BB-CD3 ζ nucleic acid sequence (SEQ ID NO: 3) and encoded amino acid sequence (SEQ ID NO: 4).
FIG. 2B depicts a lentiviral vector expressing a CAR comprising the LTG 224624P CD22ScFv-CD8TM-41BB-CD3 ζ nucleic acid sequence (SEQ ID NO: 13) and encoded amino acid sequence (SEQ ID NO: 14).
FIG. 2C depicts a lentiviral vector expressing a CAR comprising the LTG 224725P CD22ScFv-CD8TM-41BB-CD3 zeta nucleotide sequence (SEQ ID NO: 23) and the encoded amino acid sequence (SEQ ID NO: 24).
FIG. 2D depicts a lentiviral vector expressing a CAR comprising the LTG 224811 s CD22ScFv-CD8TM-41BB-CD3 ζ nucleic acid sequence (SEQ ID NO: 33) and the encoded amino acid sequence.
FIG. 2E depicts a lentiviral vector expressing a CAR comprising the LTG 224912 s CD22ScFv-CD8TM-41BB-CD3 ζ nucleic acid sequence (SEQ ID NO: 43) and the encoded amino acid sequence (SEQ ID NO: 44).
FIG. 2F depicts a lentiviral vector expressing a CAR comprising the
FIG. 2G depicts a lentiviral vector expressing a CAR comprising the LTG 220416P 16 CD22ScFv-CD8TM-41BB-CD3 ζ nucleic acid sequence (SEQ ID NO: 63) and encoded amino acid sequence (SEQ ID NO: 64).
FIG. 2H depicts a lentiviral vector expressing a CAR comprising the
FIG. 2I depicts a lentiviral vector expressing a CAR comprising the LTG 220616P 2 CD22ScFv-CD8TM-41BB-CD3 ζ nucleic acid sequence (SEQ ID NO: 83) and encoded amino acid sequence (SEQ ID NO: 84).
FIG. 2J depicts a lentiviral vector expressing a CAR comprising the
FIG. 2K depicts a lentiviral vector expressing a CAR comprising the
FIG. 2L depicts a lentiviral vector expressing a CAR comprising the LTG 220916P 17 CD22ScFv-CD8TM-41BB-CD3 ζ nucleic acid sequence (SEQ ID NO: 113) and the encoded amino acid sequence (SEQ ID NO: 114).
FIG. 2M depicts a lentiviral vector expressing a CAR comprising the LTG 221016P 20v2 CD22ScFv-CD8TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 123) and the encoded amino acid sequence (SEQ ID NO: 124).
FIG. 2N depicts a lentiviral vector expressing a CAR comprising the LTG 221616P 1CD22 ScFv-CD8TM-41BB-CD3 ζ nucleic acid sequence (SEQ ID NO: 133) and the encoded amino acid sequence (SEQ ID NO: 134).
FIG. 2O depicts a lentiviral vector expressing a CAR comprising the LTG 221716P 3v2 CD22ScFv-CD8TM-41BB-CD3 ζ nucleic acid sequence (SEQ ID NO: 143) and encoded amino acid sequence (SEQ ID NO: 144).
FIG. 2P depicts a lentiviral vector expressing a CAR comprising the
FIG. 2Q depicts a lentiviral vector expressing a CAR comprising the
FIG. 2R depicts a lentiviral vector expressing a CAR comprising the LTG 222016P 15 CD22ScFv-CD8TM-41BB-CD3 ζ nucleic acid sequence (SEQ ID NO: 173) and the encoded amino acid sequence (SEQ ID NO: 174).
Fig. 3 depicts anti-CD22 CART surface expression in primary human T cells. CAR T cells (as listed in each row of the figure) that were re-directed against the CD22 tumor antigen by using the ScFv domain were generated by lentiviral transduction with a CAR expression construct. CART detection was performed by flow cytometry. T cells were washed twice in cold PBS-EDTA buffer and stained with CD22-Fc peptide and then with anti-Fc-PE reagents. For each analysis, at least 20,000 cells were obtained. Cells were gated based on forward and side scatter, single peak discrimination, and 7AAD negativity (negotiability) so that only surviving cells were analyzed. Data were acquired on a
Figure 4 depicts anti-CD22 CAR T cells incorporating ScFv binders (16P, 16P1, 16P3v2, 16P8, 16P10, 16P13, 16P15, 16P17), utd ═ untransduced negative control, m971 ═ previously published anti-CD22 CAR positive control), which mediate cytolysis of CD 22-positive tumors in vitro. CAR T cells expressing anti-CD22 constructs were incubated overnight with either a firefly luciferase-stably transduced CD22 positive cell line (Raji and Reh) or a CD19 negative line (K562 and 293T) at effector to target ratios of 1.25, 2.5, 5, 10, 20 and 40 (x-axis). CART cytotoxic activity was then assessed by luciferase activity measurements as described in materials and methods. Each bar is the average of 3 technical replicates and the error bars represent SD. Representing at least three independent experiments.
FIG. 5 shows CD22-specific CART cell production of three cytokines (interferon- γ, TNF- α and IL-2) when co-cultured alone (middle gray, CAR only), with a CD22 positive leukemia line (Raji, black bars; Reh, light gray), or with a CD22 negative line (293T, light gray). The assay was performed overnight at a 10: 1E: T ratio, and the supernatants were then analyzed for cytokine concentration by ELISA. N ═ 3+ SEM. Negative control: untransduced T cells (utd), positive control: transduced m971CD22 CAR-T cells. The number of LTG per LV used to transduce human T cells is listed on the x-axis.
Figure 6 shows two-dimensional flow cytometry analysis of CAR expression on the surface of LV transduced T cells transduced to express no CAR (utd) or LTG2200, 2202, 2216, 2206, 2217, 2207, 2218, 2208, 2219, 2220, 2209, 2205, control GFP expression vector or control CAR-19(LTG1538) as shown by reading the rows from left to right down and listed above each figure. The y-axis dimension shows CD4 staining, and the x-axis dimension shows CAR expression by secondary staining with anti-Fc PE antibody by staining with target antigen (CD22-Fc recombinant proteins (R & D Biosystems).
Fig. 7A to 7B show cytolytic activity (CTL activity), which is the percentage of lysis of the respective luciferase-expressing target cell lines.
Figure 7A shows the CD22 positive cell lines Raji and Reh and the CD22 non-expressing line K562.
FIG. 7B shows K562-CD19 and K562-CD22 cell lines that were specifically transfected to express target antigens. For each LV transduced T cell population (as listed on the x-axis): utd (untransduced), GFP-LV, LTG1538 (anti-CD 19), m971(LTG2200, control anti-CD 22), 16p (LTG2202), 16p1(LTG2216), 16p2(LTG2206), 16p3v2(LTG2217), 16p6(LTG2207), 16p8(LTG2218), 16p10(LTG2208), 16p13(LTG2219), 16p15(LTG2220), 16p17(TG2209), 16p20(LTG2205), the ratio of the three effectors to the target was tested (E: T10: 1, 5: 1, 2.5: 1).
FIG. 8 shows the production of IFN- γ (top), IL-2 (middle) and TNF- α (bottom panel) by anti-CD22 CAR T cells after incubation overnight at a 10: 1E: T ratio with CD22 positive Raji and Reh leukemia cell lines (black or gray bars, respectively) or in the absence of target tumor cells (T cells only), and then analyzing the supernatants for cytokine concentration by ELISA. Only the negative control group of CARs was used to assess spontaneous cytokine secretion by CAR T cells. Represents at least three independent experiments. CAR-T activity was shown for non-transduced T cells (utd), GFP-lv (GFP), CD19-CAR (LTG1538), CD22 control CAR (LTG2220, m971), 16p (LTF2202), 16p1(LTG2216), 16p2(LTG2206), 16p3v2(LTG2217), 16p6(LTG2207), 16p8(LTG2218), 16p10(LTG2208), 16p13(LTG2219), 16p15(LTG2220), 16p17(LTG2209), 16p20(LTG2205) transduced T cells or leukemia targets (tumor only) incubated without T cells, as listed on the X-axis.
Figure 9 shows the ability of CAR T specific for CD22 to control disease in animal models. On day 0 of the study, immunodeficient mice (NSG) were i.v. injected with Raji leukemia cells stably expressing firefly luciferase. After injection of the luciferase substrate luciferin and imaging in the IVIS instrument imaging each animal, disease burden was measured according to the x-axis, which was reported as the mean radiance (radiance) of each group. Animals were divided into disease-burden equivalent groups of 6 mice each at
Detailed Description
Definition of
As used herein, a noun without a quantitative modification means one or more unless the context clearly dictates otherwise. For example, the term "antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen". The term "comprising" as used herein means "including". Thus, "comprising an antigen" means "including an antigen" without excluding further elements. The phrase "and/or" means "and" or ". It is also understood that any and all base sizes or amino acid sizes, as well as all molecular weights or molecular mass values given for a nucleic acid or polypeptide are approximate and provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, specific suitable methods and materials are described below. In case of conflict, the present specification, including definitions of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate a review of the various embodiments, the following terminology is provided:
the term "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is meant to encompass variations from a particular value of 20%, or in some
Unless otherwise indicated, technical terms herein are used according to conventional usage. Definitions of terms commonly used in molecular biology can be found in Benjamin Lewis, Genes VII, Oxford University Press, 1999; kendrew et al (ed), The Encyclopedia of Molecular Biology, published by Blackwell science ltd, 1994; and Robert a.meyers (ed), Molecular Biology and Biotechnology: aCompressent Desk Reference, VCH Publishers, Inc. published, 1995; and other similar references.
The present disclosure provides CD22 antibodies or fragments thereof and CARs having such CD22 antigen binding domains. The enhancement of the functional activity of the CAR is directly related to the enhancement of the functional activity of the T cell expressing the CAR. As a result of one or more of these modifications, the CARs exhibit both a high degree of cytokine-induced cytolysis and cell surface expression on transduced T cells, as well as increased levels of T cell expansion in vivo and persistence of transduced CAR-expressing T cells.
The unique ability to combine functional portions derived from different protein domains is a key innovative feature of CARs. The choice of each of these protein domains is a key design feature, as are the ways of their specific combinations. Each design domain is an essential component that can be used between different CAR platforms to engineer the function of lymphocytes. For example, the extracellular binding domain can be selected such that a CAR that is otherwise ineffective becomes effective.
The constant framework components of the immunoglobulin-derived protein sequence used to establish the extracellular antigen-binding domain of the CAR may be completely neutral or it may self-associate and drive T cells to a state of metabolic failure, thereby making therapeutic T cells expressing the CAR far less effective. This occurs independently of the antigen binding function of the CAR domain. In addition, the selection of intracellular signaling domains may also control the activity and persistence of the therapeutic lymphocyte population for immunotherapy. While the ability to bind a target antigen and the ability to transmit an activation signal to T cells via these extracellular and intracellular domains, respectively, is an important CAR design aspect, it has also become apparent that the choice of the source of the extracellular antigen-binding fragment can have a significant impact on the efficacy of the CAR and thus have a defined effect on the function and clinical utility of the CAR.
Surprisingly and unexpectedly, it has now been found that the functional activity of T cells expressing CARs can also be determined using fully human antigen binding domains in the CARs rather than using mouse-derived antigen binding fragments that are prone to induce anti-mouse immune responses and CAR T elimination in the host (see, the upenn-specific clinical three using mouse derived SS1 ScFv sequence, NCT 02159716).
The CARs disclosed herein are expressed at high levels in a cell. The CAR-expressing cells have a high rate of proliferation in vivo, produce large amounts of cytokines, and have high cytotoxic activity against cells that have CD22 antigen bound to the CAR on their surface. The use of the human extracellular CD22 antigen binding domain results in the production of CARs that function better in vivo, while avoiding the induction of anti-CAR immunity and killing of CAR T cell populations in the host immune response. CARs expressing fully human extracellular CD22ScFv antigen binding domains exhibit excellent activity/properties, including i) protection against poor CAR T persistence and function as seen in the case of mouse-derived binding sequences; ii) to effect a lack of regional (i.e., intra-pleural) delivery of the CAR; and iii) the ability to generate CAR T cell designs based on both high and low affinity binders to CD 19. The latter property allows researchers to better modulate the efficacy and toxicity, and/or tissue specificity, of CAR T products, since lower affinity binders may have higher specificity for tumors than normal tissues, due to the expression of CD22 on tumors being higher than normal tissues, which may prevent off-target off tumor toxicity (on-target off tumor toxicity) and bystander (bystander) cell killing.
The following is a detailed description of the CARs of the invention, including a description of their extracellular CD22 antigen binding domain, transmembrane domain, and intracellular domain, as well as additional descriptions of CARs, antibodies and antigen binding fragments, conjugates, nucleotides, expressions, vectors, and host cells, methods of treatment, compositions, and kits for using the disclosed CARs.
A. Chimeric Antigen Receptor (CAR)
The CARs disclosed herein comprise at least one CD22 antigen binding domain capable of binding to CD22, at least one transmembrane domain, and at least one intracellular domain.
Chimeric Antigen Receptors (CARs) are artificially constructed hybrid proteins or polypeptides comprising an antigen binding domain (e.g., a single chain variable fragment (scFv)) of an antibody linked to a T cell signaling domain via a transmembrane domain. Features of the CARs include their ability to redirect T cell specificity and reactivity toward selected targets in a non-Major Histocompatibility Complex (MHC) restricted manner and using the antigen binding properties of monoclonal antibodies. non-MHC restricted antigen recognition confers CAR-expressing T cells the ability to recognize antigen independently of antigen processing, thereby bypassing the major mechanism of tumor escape. Furthermore, when expressed in T cells, the CARs advantageously do not dimerize with endogenous T Cell Receptor (TCR) alpha and beta chains.
As disclosed herein, the intracellular T cell signaling domain of the CAR can comprise, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain refers to the portion of the CAR that comprises the intracellular domain of the T cell receptor, such as, but not limited to, the intracellular portion of the CD3 zeta protein. A costimulatory signaling domain refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule, a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of lymphocytes to an antigen.
1. Extracellular domain
In one embodiment, the CAR comprises a target-specific binding member, which is also referred to as an antigen-binding domain or portion. The choice of domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen binding domain can be selected to recognize ligands that serve as cell surface markers on target cells associated with a particular disease state. Thus, some examples of cell surface markers that can serve as ligands for the antigen binding domain in a CAR include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.
In one embodiment, the CAR can be engineered to target a tumor antigen of interest by engineering a desired antigen binding domain that specifically binds to the antigen on the tumor cell. Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. The choice of antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β -human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), enterocarboxyesterase, mut hsp70-2, M-CSF, prostatase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2/neu, leptin and telomerase, prostate cancer tumor antigen-1 (pro-carcinoma mordant antigen-1, PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin 19, insulin growth factor (IGF-I, IGF), IGF-growth factor I, IGF, IGF-II, IGF-I receptor and CD 22. The tumor antigens disclosed herein are included by way of example only. This list is not intended to be exclusive and other examples will be apparent to those skilled in the art.
In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignancy. Malignant tumors express a variety of proteins that can serve as target antigens for immune challenge. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase and GP100 in melanoma, and Prostate Acid Phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, for example the oncogene HER-2/Neu/ErbB-2. Another group of target antigens are cancer-embryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute a true tumor-specific immunoglobulin antigen that is characteristic of an individual's tumor. B cell differentiation antigens (e.g., CD19, CD20, CD22, BCMA, ROR1, and CD37) are further candidates as target antigens in B cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, CD22, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, but with limited success.
In a preferred embodiment, the tumor antigen is CD22, and the tumor associated with CD22 expression comprises mesothelioma, ovarian, and pancreatic cancer of the lung, or any combination thereof, that express high levels of the extracellular protein CD 22.
The type of tumor antigen can also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is unique to tumor cells and does not appear on other cells in the body. TAAs are not unique to tumor cells and are instead expressed on normal cells under conditions that fail to induce an immune-tolerant state against the antigen. Expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAA may be an antigen expressed on normal cells during fetal development when the immune system is immature and unable to respond, or it may be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.
Some non-limiting examples of TSA or TAA include the following: differentiation antigens such as MART-1/Melana (MART-I), gp100(Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor specific multiple lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p 15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as EBVA, an Epstein Barr virus (Epstein Barr virus) antigen, and Human Papilloma Virus (HPV) antigens E6 and E7. Other protein-based macroantigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, P185erbB2, P180erbB-3, C-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, P15, P16, 43-9F, 5T4, 791Tgp72, alpha fetoprotein, beta-HCG, BCA225, BTA, CA 125, CA 15-3\ CA 27.29 BCAA, CA 195, CA 242, CA-50, CAM 5, CD68\ P1, CO-029, G-5, G250, Ga733\ CAM, gp 175-63344, M-6350, MOV-25, SDC 7, SDC-24, GCA-GCGCTC55, GCGCGCGCGCTC9, GCGCTC9, GCTC9, GCTCK-3670, GCATG-24, GCA-3690, GCAS-36387, GCA-binding protein, GCS-24, GCAS-3690, GCA-binding protein, and GCS-6, TAAL6, TAG72, TLP and TPS.
In one embodiment, the antigen binding domain portion of the CAR targets an antigen including, but not limited to: CD19, CD20, CD22, ROR1, CD33, CD38, CD123, CD138, BCMA, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, FGFR4, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, and the like.
In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD22 antigen.
In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22scFv1 antigen binding domain comprises SEQ ID NO: 1, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence identical to SEQ ID NO: 2, has 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 scFv2 antigen binding domain comprises SEQ ID NO: 11, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence identical to SEQ ID NO: 12 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22 ScFv3 antigen binding domain comprises SEQ ID NO: 21, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD22 ScFv3 antigen binding domain comprises SEQ ID NO: 22, or an amino acid sequence identical to SEQ ID NO: 22 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22 ScFv4 antigen binding domain comprises SEQ ID NO: 31, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD22 ScFv4 antigen binding domain comprises SEQ ID NO: 32, or an amino acid sequence identical to SEQ ID NO: 32, has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22 ScFv5 antigen binding domain comprises SEQ ID NO: 41, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD22 ScFv5 antigen binding domain comprises SEQ ID NO: 42, or an amino acid sequence identical to SEQ ID NO: 42 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22 ScFv6 antigen binding domain comprises SEQ ID NO: 51, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD22 ScFv6 antigen binding domain comprises SEQ ID NO: 52, or an amino acid sequence identical to SEQ ID NO: 52 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22 ScFv7 antigen binding domain comprises SEQ ID NO: 61, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD22 ScFv7 antigen binding domain comprises SEQ ID NO: 62, or an amino acid sequence identical to SEQ ID NO: 62 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22 ScFv8 antigen binding domain comprises SEQ ID NO: 71, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD22 ScFv8 antigen binding domain comprises SEQ ID NO: 72, or an amino acid sequence identical to SEQ ID NO: 72 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22 ScFv9 antigen binding domain comprises SEQ ID NO: 81, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD22 ScFv9 antigen binding domain comprises SEQ ID NO: 82, or an amino acid sequence identical to SEQ ID NO: 82, has 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22ScFv10 antigen binding domain comprises SEQ ID NO: 91, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22ScFv10 antigen binding domain comprises SEQ ID NO: 92, or an amino acid sequence identical to SEQ ID NO: 92 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22ScFv11 antigen binding domain comprises SEQ ID NO: 101, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD22ScFv102 antigen binding domain comprises SEQ ID NO: 92, or an amino acid sequence identical to SEQ ID NO: 102 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22 ScFv12 antigen binding domain comprises SEQ ID NO: 111, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22ScFv112 antigen binding domain comprises SEQ ID NO: 92, or an amino acid sequence identical to SEQ ID NO: 112 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22ScFv13 antigen binding domain comprises SEQ ID NO: 121, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22ScFv13 antigen binding domain comprises SEQ ID NO: 122, or an amino acid sequence identical to SEQ ID NO: 122 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22ScFv14 antigen binding domain comprises SEQ ID NO: 131, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22ScFv14 antigen binding domain comprises SEQ ID NO: 132, or an amino acid sequence identical to SEQ ID NO: 132 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22ScFv15 antigen binding domain comprises SEQ ID NO: 141, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22ScFv15 antigen binding domain comprises SEQ ID NO: 142, or an amino acid sequence identical to SEQ ID NO: 142 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22ScFv16 antigen binding domain comprises SEQ ID NO: 151, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22ScFv16 antigen binding domain comprises SEQ ID NO: 152, or an amino acid sequence identical to SEQ ID NO: 152 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22ScFv17 antigen binding domain comprises SEQ ID NO: 161, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22ScFv17 antigen binding domain comprises SEQ ID NO: 162, or an amino acid sequence identical to SEQ ID NO: 162 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
In a preferred embodiment, the isolated nucleic acid molecule encoding an extracellular CD22ScFv18 antigen binding domain comprises SEQ ID NO: 171, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD22ScFv18 antigen binding domain comprises SEQ ID NO: 172, or an amino acid sequence identical to SEQ ID NO: 172 has an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv1, as a combination to produce the light chain binding characteristics of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce the heavy chain binding characteristics of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce the binding characteristics of a CD22-specific scFv1, by co-expressing in a single scFv amino acid sequence of SEQ ID NOs: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 10.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv2, as a combination to produce a light chain binding characteristic of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce a heavy chain binding characteristic of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce a binding characteristic of a CD22-specific scFv2, by co-expressing in a single amino acid sequence of SEQ ID NOs: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19 and SEQ ID NO: 20, and (3) performing.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv3, as a combination to produce a light chain binding characteristic of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce a heavy chain binding characteristic of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce a binding characteristic of a CD22-specific scFv3, by co-expressing in a single amino acid sequence of SEQ ID NOs: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29 and SEQ ID NO: 30, and (3) carrying out.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively) each independently contribute to the binding characteristics of the CD22-specific scFv4, as a combination to produce the light chain binding characteristics of the scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce the heavy chain binding characteristics of the scFv (HCDR1+ HCDR2+ HCDR3), and as a combination of six IDs, as a combination to together produce the binding characteristics of the CD22-specific scFv4, as a group, by co-expressing in a single amino acid sequence the amino acid sequences of SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39 and SEQ ID NO: 40.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv5, as a combination to produce the light chain binding characteristics of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce the heavy chain binding characteristics of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce the binding characteristics of a CD22-specific scFv5, by co-expressing in a single amino acid sequence of SEQ ID NOs: 45. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48. SEQ ID NO: 49 and SEQ ID NO: 50, the reaction is carried out.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv6, as a combination to produce a light chain binding characteristic of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce a heavy chain binding characteristic of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce a binding characteristic of a CD22-specific scFv6, by co-expressing in a single amino acid sequence of SEQ ID NOs: 55. SEQ ID NO: 56. SEQ ID NO: 57. SEQ ID NO: 58. SEQ ID NO: 59 and SEQ ID NO: 60, the process is carried out.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv7, as a combination to produce the light chain binding characteristics of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce the heavy chain binding characteristics of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce the binding characteristics of a CD22-specific scFv7, by co-expressing in a single amino acid sequence of SEQ ID NOs: 65. SEQ ID NO: 66. SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69 and SEQ ID NO: 70.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv8, as a combination to produce a light chain binding characteristic of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce a heavy chain binding characteristic of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce a binding characteristic of a CD22-specific scFv8, by co-expressing in a single amino acid sequence of SEQ ID NOs: 75. SEQ ID NO: 76. SEQ ID NO: 77. SEQ ID NO: 78. SEQ ID NO: 79 and SEQ ID NO: 80.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv9, as a combination to produce a light chain binding characteristic of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce a heavy chain binding characteristic of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce a binding characteristic of a CD22-specific scFv9, by co-expressing in a single amino acid sequence of SEQ ID NOs: 85. SEQ ID NO: 86. SEQ ID NO: 87. SEQ ID NO: 88. SEQ ID NO: 89 and SEQ ID NO: 90, and (3) carrying out.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv10, as a combination to produce a light chain binding characteristic of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce a heavy chain binding characteristic of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce a binding characteristic of a CD22-specific scFv10, by co-expressing in a single amino acid sequence of SEQ ID NOs: 95. SEQ ID NO: 96. SEQ ID NO: 97. SEQ ID NO: 98. SEQ ID NO: 99 and SEQ ID NO: 100, respectively.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv11, as a combination to produce the light chain binding characteristics of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce the heavy chain binding characteristics of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce the binding characteristics of a CD22-specific scFv11, by co-expressing in a single amino acid sequence of SEQ ID NOs: 105. SEQ ID NO: 106. SEQ ID NO: 107. SEQ ID NO: 108. SEQ ID NO: 109 and SEQ ID NO: 110.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv12, as a combination to produce the light chain binding characteristics of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce the heavy chain binding characteristics of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce the binding characteristics of a CD22-specific scFv12, by co-expressing in a single amino acid sequence of SEQ ID NOs: 115. SEQ ID NO: 116. SEQ ID NO: 117. SEQ ID NO: 118. SEQ ID NO: 119 and SEQ ID NO: 120, are carried out.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 128, SEQ ID NO: 129, and SEQ ID NO: 130, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv13, as a combination to produce the light chain binding characteristics of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce the heavy chain binding characteristics of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce the binding characteristics of a CD22-specific scFv13, by co-expressing in a single amino acid sequence of SEQ ID NOs: 125. SEQ ID NO: 126. SEQ ID NO: 127. SEQ ID NO: 128. SEQ ID NO: 129 and SEQ ID NO: 130.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 135, SEQ ID NO: 136, and SEQ ID NO: 137, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 138, SEQ ID NO: 139, and SEQ ID NO: 140, respectively) each independently contribute to the binding characteristics of a CD22-specific scFv14, as a combination to produce the light chain binding characteristics of a scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce the heavy chain binding characteristics of a scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six IDs, as a combination together to produce the binding characteristics of a CD22-specific scFv14, by co-expressing in a single amino acid sequence of SEQ ID NOs: 135. SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 and SEQ ID NO: 140.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150, respectively) each independently contribute to the binding characteristics of the CD22-specific scFv15, as a combination to produce the light chain binding characteristics of the scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce the heavy chain binding characteristics of the scFv (HCDR1+ HCDR2+ HCDR3), and as a combination of six IDs, as a combination together to produce the binding characteristics of the CD22-specific scFv15, as a group, by co-expressing in a single amino acid sequence the amino acid sequences of SEQ ID NO: 145. SEQ ID NO: 146. SEQ ID NO: 147. SEQ ID NO: 148. SEQ ID NO: 149 and SEQ ID NO: 150.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 155, SEQ ID NO: 156, and SEQ ID NO: 157, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160, respectively) each independently contribute to the binding characteristics of the CD22-specific scFv16, as a combination to the light chain binding characteristics of the scFv (LCDR1+ LCDR2+ LCDR3), as a combination to the heavy chain binding characteristics of the scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six SEQ IDs, as a combination to together the binding characteristics of the CD22-specific scFv16, by co-expressing in a single amino acid sequence the amino acid sequences of SEQ ID NO: 155. SEQ ID NO: 156. SEQ ID NO: 157. SEQ ID NO: 158. SEQ ID NO: 159 and SEQ ID NO: 160.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 165, SEQ ID NO: 166 and SEQ ID NO: 167, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 168, SEQ ID NO: 169 and SEQ ID NO: 170, respectively) each independently contribute to the binding characteristics of the CD22-specific scFv17, as a combination to the light chain binding characteristics of the scFv (LCDR1+ LCDR2+ LCDR3), as a combination to the heavy chain binding characteristics of the scFv (HCDR1+ HCDR2+ HCDR 8242), and as a combination of six SEQ IDs, as a combination to together the binding characteristics of the CD22-specific scFv17, by co-expressing in a single amino acid sequence the amino acid sequences of SEQ ID NO: 165. SEQ ID NO: 166. SEQ ID NO: 167. SEQ ID NO: 168. SEQ ID NO: 169 and SEQ ID NO: 170.
In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, identified as SEQ ID NO: 175, SEQ ID NO: 176 and SEQ ID NO: 177, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 178, SEQ ID NO: 179 and SEQ ID NO: 180, respectively) each independently contribute to the binding characteristics of CD22-specific scFv18, as a combination to produce the light chain binding characteristics of scFv (LCDR1+ LCDR2+ LCDR3), as a combination to produce the heavy chain binding characteristics of scFv (HCDR1+ HCDR2+ HCDR3) and as a combination of six SEQ IDs, as a combination to together to produce the binding characteristics of CD22-specific scFv18, by co-expressing in a single amino acid sequence the amino acid sequences of SEQ ID NO: 175. SEQ ID NO: 176. SEQ ID NO: 177. SEQ ID NO: 178. SEQ ID NO: 179 and SEQ ID NO: 180, respectively.
In various embodiments of the CD22-specific CARs disclosed herein, the general scheme is shown in figure 1 and includes, from N-terminus to C-terminus, a signal or leader peptide, an anti-CD22 ScFv, an extracellular linker, CD8 across a membrane, 4-1BB, CD3 ζ, where the cloning sites for the linking domains are indicated in bold letters.
In one embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 3 and encodes a nucleic acid sequence comprising SEQ ID NO: 4 [ LTG2202 LP-16P-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2A) ].
In one embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 3 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 4 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [ LTG2202 LP-16P-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2A) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 13 and encodes a nucleic acid sequence comprising SEQ ID NO: 14 [ LTG2246 LP-24P-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2B) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 13 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 14 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [ LTG2246 LP-24P-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2B) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 23 and encodes a nucleic acid sequence comprising SEQ ID NO: 24 [ LTG2247 LP-25P-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (as shown in figure 2C) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 23 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 24 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2247 LP-25P-CD 8TM-41BB-CD3 ζ CAR amino acid sequence (as shown in figure 2C) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 33 and encodes a nucleic acid sequence comprising SEQ ID NO: 34 [ LTG2248 LP-11S-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2D) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 33 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 34 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [ LTG2248 LP-11S-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2D) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 43 and encodes a nucleic acid sequence comprising SEQ ID NO: 44 [ LTG2249 LP-12S-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in FIG. 2E) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 43 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 44 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [ LTG2249 LP-12S-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2E) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 53 and encodes a nucleic acid sequence comprising SEQ ID NO: 54 [ (LTG2203 LP-16P3-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2F) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 53 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 54 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2203 LP-16P3-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2F) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 63, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 64 [ LTG2204 LP-16P16-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in FIG. 2G) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 63 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 64 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2204 LP-16P16-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2G) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 73 and encodes a nucleic acid sequence comprising SEQ ID NO: 74 [ LTG2205 LP-16P20-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2H) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 73 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 74 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2205 LP-16P20-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2H) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 83 and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 84 [ LTG2206 LP-16P2-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in FIG. 2I) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 83 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 84 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2206 LP-16P2-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2I) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 93, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 94 [ LTG2207 LP-16P6-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in FIG. 2J) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 93 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 94 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2207 LP-16P6-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2J) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 103, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 104 [ LTG2208 LP-16P10-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2K) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 103 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 104 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2208 LP-16P10-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2K) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 113 and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 114 [ LTG2209 LP-16P17-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2L) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 113 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 114 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [ LTG2209 LP-16P17-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2L) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 123 and encodes a nucleic acid sequence comprising SEQ ID NO: 124 [ LTG2210 LP-16P20v2-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in FIG. 2M) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 123 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 124 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2210 LP-16P20v2-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2M) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 133 and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 134 [ LTG2216 LP-16P1-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2N) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 133 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 134 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2216 LP-16P1-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2N) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 143 and encodes a nucleic acid sequence comprising SEQ ID NO: 144 [ LTG2217 LP-16P3v2-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2O) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 143 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 144 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2217 LP-16P17-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2O) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 153 and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 154 [ LTG2218 LP-16P8-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in FIG. 2P) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 153 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 154, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [ LTG2218 LP-16P8-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2P) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 163 and encodes a nucleic acid sequence comprising SEQ ID NO: 164 [ LTG2219 LP-16P13-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in FIG. 2Q) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 163 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 164 or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [ LTG2219 LP-16P13-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2Q) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 173, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 174 [ LTG2220 LP-16P15-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2R) ].
In another embodiment, the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 173, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a polypeptide comprising the nucleic acid sequence of SEQ ID NO: 174, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [ LTG2220 LP-16P15-CD 8TM-41BB-CD3 ζ amino acid sequence (as shown in figure 2R) ].
Surface expression of anti-CD22 CARs incorporating single chain variable fragment (ScFv) sequences reactive with CD22 antigen is shown in example 2 below and summarized in table 2, table 3 and table 6. The expression level of each ScFv-containing CAR was determined by flow cytometry analysis of LV transduced T cells from healthy donors using recombinant CD22-Fc peptide followed by anti-human Fc F (ab') 2 fragment conjugated to PE, and detected by flow cytometry (see figure 6). ScFv-based anti-CD22 CAR constructs LTG2202, LTG2216, LTG2217, LTG2218, LTG2208, LTG2219, LTG2220, and LTG2209 were highly expressed in human primary T cells (as shown by the gated population) compared to the untransduced T cell control (non-gated population of cells, UTD). Representative results from one donor are shown.
As shown in example 2 and fig. 4, 7A and 7B, the high cytolytic activity of CD22 CARs was shown when Lentiviral Vectors (LV) expressing the following CARs were generated and tested for anti-leukemic activity. Each experimental CAR comprises the 4-1BB/CD 3-zeta chain signaling motif and the specific anti-CD22 binding motif/domain indicated herein. Leukemia target lines with different surface expression of CD22 were used: raji and Reh; and CD19 negative K562 and 293T. ScFv-based anti-CD22 CAR constructs LTG2202, LTG2216, LTG2217, LTG2218, LTG2208, LTG2219, LTG220 and LTG2209 (expressing ScFv1(16P), ScFv2(16P1), ScFv3(16P3v2), ScFv3(16P3v2), ScFv4(16P8), ScFv5(16P10), ScFv6(16P13), ScFv7(16P15), ScFv8(16P17), respectively) were able to efficiently cleave CD 22-high tumor lines Raji and Reh, whereas the specific cleavage activity of these CAR constructs was low or absent for K562 or 293T (see figures 4, 7A, 7B). These results indicate the efficiency and specificity of the CAR constructs produced.
anti-CD22 CAR T cells were then evaluated for their cytokine secretion capacity. Tumor cells were incubated overnight with CAR T cells or control T cells at a 10: 1 effector to target ratio and culture supernatants were analyzed for IFN γ, TNF α, and IL-2 by ELISA (see, fig. 8). Notably, CAR T cells transduced with LTG2202, LTG2216, LTG2217, LTG2218, LTG2208, LTG2219, LTG2220, and LTG2209 (expressing scFv1(16P), scFv2(16P1), scFv3(16P3v2), scFv3(16P3v2), scFv4(16P8), scFv5(16P10), scFv6(16P13), scFv7(16P15), scFv8(16P17), respectively) produced high levels of IFN γ, whereas the negative control (untransduced, utd) did not produce significant cytokine induction. However, significant differences were observed in TNF- α and IL-2 production. Surprisingly, CD22CAR LTG2202 produced significantly lower levels of TNF-a and IL-2 against Reh tumor lines, and each vector had a different ability to produce IL-2 and TNF-a against the tumor line targets tested. These differences will lead to different anti-tumor and toxicity profiles and will be implemented individually depending on the disease burden in a specific disease context. CAR (m971) used as a positive control was used to benchmark results as it is currently in clinical trials and so far safe to use in a late stage disease setting.
Without intending to be limited to any particular mechanism of action, it is believed that possible causes of enhanced therapeutic function associated with the exemplary CARs of the invention include, for example and without limitation: a) improved lateral movement within the plasma membrane to allow more efficient signal transduction, b) superior localization within the plasma membrane microdomains (e.g., lipid rafts), and greater ability to interact with transmembrane signaling cascades associated with T cell activation, c) superior localization within the plasma membrane by preferentially moving away from inhibitory (dampening) or down-regulatory interactions (e.g., less proximity or interaction with phosphatases (e.g., CD45), and d) superior assembly into T cell receptor signaling complexes (i.e., immune synapses), or any combination thereof.
While the present disclosure has been exemplified with exemplary extracellular only CD22 variable heavy chains and ScFv antigen binding domains, other nucleotide and/or amino acid variants within CD22 variable heavy chains and ScFv antigen binding domains can be used to derive the CD22 antigen binding domains for use in the CARs described herein.
Depending on the desired antigen to be targeted, the CAR may additionally be engineered to include a suitable antigen binding domain specific for the desired antigen target. For example, if CD22 is the desired antigen to be targeted, an antibody directed against CD22 can be used as the antigen binding domain incorporated into the CAR.
In an exemplary embodiment, the antigen binding domain portion of the CAR is additionally targeted to
In an exemplary embodiment, the antigen binding domain portion of the CAR additionally targets mesothelin. Preferably, the antigen binding domain in the CAR is an anti-mesothelin ScFv, wherein the nucleic acid sequence of the anti-mesothelin ScFv comprises the amino acid sequence of SEQ id no: 198 to seq id no. In one embodiment, the anti-mesothelin ScFv comprises a nucleic acid sequence encoding SEQ ID NO: 199, or a nucleic acid sequence of an amino acid sequence of seq id no. In another embodiment, the anti-mesothelin ScFv portion of the CAR comprises SEQ ID NO: 199, or a pharmaceutically acceptable salt thereof. In another exemplary embodiment, the nucleic acid sequence of the CAR expressing an anti-mesothelin ScFv consists of seq id: 200. In another embodiment, the amino acid sequence of anti-mesothelin CAR LTG1904 is SEQ ID NO: shown at 201.
In one aspect of the invention, there is provided a CAR capable of binding to a non-TSA or non-TAA, including antigens such as but not limited to those derived from: the family of retroviridae (e.g., human immunodeficiency viruses such as HIV-1 and HIV-LP), the family of picornaviridae (e.g., poliovirus, hepatitis a virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis b virus, parvovirus, adenoviridae, the family of herpesviridae [ e.g., herpes
In another aspect of the invention, there is provided a CAR capable of binding to an antigen derived from the following bacterial strains: staphylococcus (Staphyloccci), Streptococcus (Streptococcus), Escherichia coli (Escherichia coli), Pseudomonas (Pseudomonas) or Salmonella (Salmonella). In particular, there is provided a CAR capable of binding to an antigen derived from, for example, an infectious bacterium such as: helicobacter pylori (Helicobacter pylori), Legionella pneumophila (Legionella pneumophila), bacterial strains of mycobacterium (mycobacterium sp) (e.g. mycobacterium tuberculosis (m.tuberculosis), mycobacterium avium (m.avium), mycobacterium intracellulare (m.intracellularis), mycobacterium kansasii (m.kansaii) or mycobacterium gordoniae (m.gordonia)), Staphylococcus aureus (Staphylococcus aureus), Neisseria gonorrhoeae (Neisseria gordoniae), Neisseria meningitidis (neuroserinia meningitidis), Listeria monocytogenes (Listeria monocytogenes), Streptococcus pyogenes (Streptococcus pyogenes), Group a Streptococcus (Group a), Group B Streptococcus (Streptococcus pneumoniae) (Clostridium Streptococcus pneumoniae)), Streptococcus pneumoniae (Clostridium pneumoniae), or Streptococcus pneumoniae (Clostridium Streptococcus pneumoniae), or combinations thereof.
2. Transmembrane domain
With respect to the transmembrane domain, the CAR comprises one or more transmembrane domains fused to the extracellular CD22 antigen binding domain of the CAR.
The transmembrane domain may be derived from natural sources or synthetic sources. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
The transmembrane regions particularly useful in the CARs described herein may be derived from (i.e. comprise at least the following transmembrane regions): an α, β, or zeta chain of a T cell receptor, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF 19. Alternatively, the transmembrane domain may be synthetic, in which case it will contain predominantly hydrophobic residues, such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be present at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) may form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine diads provide particularly suitable linkers.
In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used in addition to the transmembrane domains described above.
In some cases, transmembrane domains may be selected or substituted with amino acids to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interaction with other members of the receptor complex.
In one embodiment, the transmembrane domain in the CAR of the invention is the CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises SEQ ID NO: 181. In one embodiment, the CD8 transmembrane domain comprises a sequence encoding SEQ ID NO: 182, or a nucleic acid sequence of the amino acid sequence of seq id no. In another embodiment, the CD8 transmembrane domain comprises SEQ ID NO: 182, or a pharmaceutically acceptable salt thereof.
In one embodiment, the encoded transmembrane domain comprises SEQ ID NO: 182 having at least one, two, or three modifications (e.g., substitutions) but NO more than 20, 10, or 5 modifications (e.g., substitutions), or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 182 has a sequence identity of 95% to 99%.
In some cases, the transmembrane domain of the CAR comprises a cd8.α. In one embodiment, the CD8 hinge domain comprises SEQ ID NO: 183. In one embodiment, the CD8 hinge domain comprises a nucleotide sequence encoding SEQ ID NO: 184, or a nucleotide sequence of the amino acid sequence of seq id no. In another embodiment, the CD8 hinge domain comprises seq id NO: 184, or a sequence having 95% to 99% identity thereto.
In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.
3. Spacer domains
In a CAR, the spacer domain can be disposed between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. Spacer domain means any oligopeptide or polypeptide used to link the transmembrane domain to the extracellular domain and/or to link the transmembrane domain to the intracellular domain. The spacer domain comprises up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.
In some embodiments, the linker may comprise a spacer element, which when present, increases the size of the linker such that the distance between the effector molecule or detectable label and the antibody or antigen binding fragment increases. Exemplary spacers are known to those of ordinary skill and include those listed in: U.S. patent nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, and U.S. patent publication nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.
The spacer domain preferably has a sequence that promotes binding of the CAR to the antigen and enhances signal conduction into the cell. Some examples of amino acids that are expected to facilitate binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can be used as the amino acids that make up the spacer domain.
As spacer domains, all or part of amino acids 137 to 206 of the hinge region of CD8. alpha. (SEQ ID NO: 39) (NCBI RefSeq: NP. sub. - -001759.3), amino acids 135 to 195 of CD8. beta. (GenBank: AAA35664.1), amino acids 315 to 396 of CD4 (NCBI RefSeq: NP. sub. - -000607.1) or amino acids 137 to 152 of CD28 (NCBI RefSeq: NP. sub. - -006130.1) can be used. In addition, as the spacer domain, a part of the constant region of the antibody H chain or L chain can be used. In addition, the spacer domain may be a synthetic sequence.
In addition, in CAR, the signal peptide sequence may be linked to the N-terminus. The signal peptide sequence is present at the N-terminus of many secreted and membrane proteins and is 15 to 30 amino acids in length. Since many of the protein molecules mentioned above as intracellular domains have signal peptide sequences, these signal peptides can be used as signal peptides for CARs. In one embodiment, the signal peptide comprises SEQ ID NO: 191, or a pharmaceutically acceptable salt thereof.
4. Intracellular domains
The cytoplasmic domain or in other cases the intracellular signaling domain of the CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can generally be used, in many cases the entire chain need not be used. To the extent that truncated portions of intracellular signaling domains are used, such truncated portions may be used in place of the entire chain, so long as they transduce effector functional signals. The term intracellular signaling domain is therefore meant to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.
Some preferred examples of intracellular signaling domains for use in a CAR include T Cell Receptors (TCRs) and cytoplasmic sequences of co-receptors that act synergistically to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequences with the same functional capacity.
It is known that the signal generated by the TCR alone is not sufficient to fully activate T cells and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be thought of as being mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).
The primary cytoplasmic signaling sequence modulates primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary cytoplasmic signaling sequence that functions in a stimulatory manner may comprise signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.
Some examples of primary cytoplasmic signaling sequences comprising ITAMs that are particularly useful for the CARs disclosed herein include those derived from TCR ζ (CD3 ζ), FcR γ, FcR β, CD3 γ, CD3, CD3, CD5, CD22, CD79a, CD79b, and CD66 d. Specific non-limiting examples of ITAMs include peptides having the following sequences: amino acids 51 to 164 of CD3.ζ. (NCBIRefSeq: NP. sub. - -932170.1), amino acids 45 to 86 of Fc.. RI.. gamma. (NCBI RefSeq: NP. sub. - -004097.1), amino acids 201 to 244 of Fc.. RI.. beta. - - (NCBI RefSeq: NP. sub. - -000130.1), amino acids 139 to 182 of CD3. gamma. (NCBI RefSeq: NP. sub. - -000064.1), amino acids 128 to 171 of CD3. (NCBI RefSeq: NP. sub. - -000723.1),
In a preferred embodiment, the intracellular domain of the CAR can be designed to comprise the CD 3-zeta signaling domain alone or in combination with any other desired cytoplasmic domain that can be used in the CAR context. For example, the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. A costimulatory signaling region refers to the portion of the CAR that comprises the intracellular domain of the costimulatory molecule. Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that are required for an effective response of lymphocytes to an antigen. Some examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, and the like. Some specific non-limiting examples of such co-stimulatory molecules include peptides having the following sequences: amino acids 236 to 351 of CD2 (NCBI RefSeq: np.sub. - -001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq: np.sub. - -000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: np.sub. - -055022.2), amino acids 207 to 235 of CD8.α (NCBI RefSeq: np.sub. - -001759.3), amino acids 196 to 210 of CD83 (GenBank: AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq: np.sub. - -006130.1), amino acids 214 to 255 of CD137(4-1BB, NCBI RefSeq: np.sub. - -001552.2), amino acids 241 to 277 of CD134 (RefSeq: 40, NCBI ref: seq: np. 003318.1) and amino acids 166 to 199-036224.1 of CD134 have the same function as these amino acids, and their peptides 59np.5925. Thus, although the disclosure herein is primarily exemplified with 4-1BB as the co-stimulatory signaling element, other co-stimulatory elements are also within the scope of the present disclosure.
The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in random or a specific order. Optionally, short oligopeptide linkers or polypeptide linkers (preferably 2 to 10 amino acids in length) may form the linkage. Glycine-serine diads provide particularly suitable linkers.
In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD 3-zeta and the signaling domain of
In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD 3-zeta, wherein the signaling domain of 4-1BB comprises SEQ ID NO: 186 and the signaling domain of CD 3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 188.
In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD 3-zeta, wherein the signaling domain of 4-1BB comprises a sequence encoding SEQ ID NO: 187 and the signaling domain of CD 3-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 189, or a nucleic acid sequence of the amino acid sequence of 189.
In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD 3-zeta, wherein the signaling domain of 4-1BB comprises SEQ ID NO: 187 and the signaling domain of CD 3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 189 as shown in seq id no.
Additional description of CAR
Also specifically included within the scope of the invention are functional portions of the CARs disclosed herein. The term "functional portion" when used with reference to a CAR refers to any portion or fragment of one or more CARs disclosed herein that retains the biological activity of the CAR of which it is a part (the parent CAR). Functional portions encompass, for example, those portions of the CAR that retain the ability to recognize a target cell or detect, treat, or prevent a disease to a similar extent, to the same extent, or to a greater extent, as compared to the parent CAR. With reference to the parent CAR, the functional portion can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.
The functional moiety may comprise an additional amino acid at the amino or carboxy terminus of the moiety or at both termini, which additional amino acid is not present in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional moiety, e.g., recognizing target cells, detecting cancer, treating or preventing cancer, and the like. More desirably, the additional amino acids enhance the biological activity as compared to the biological activity of the parent CAR.
Included within the scope of the present disclosure are functional variants of the CARs disclosed herein. The term "functional variant" as used herein refers to a CAR, polypeptide, or protein having significant or significant sequence identity or similarity to a parent CAR, which functional variant retains the biological activity of the CAR (which functional variant is a variant thereof). Functional variants encompass, for example, those variants of the CAR described herein (parent CAR) that retain the ability to recognize the target cell to a similar extent, to the same extent, or to a greater extent, as compared to the parent CAR. With reference to a parent CAR, a functional variant can, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98% or more identity in amino acid sequence to the parent CAR.
A functional variant may, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant may comprise the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent CAR.
The amino acid substitution of the CAR is preferably a conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which an amino acid having a particular physical and/or chemical property is exchanged for another amino acid having the same or similar chemical or physical property. For example, a conservative amino acid substitution can be an acidic/negatively charged polar amino acid in place of another acidic/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a non-polar side chain in place of another amino acid having a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic/positively charged polar amino acid in place of another basic/positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid having a polar side chain in place of another uncharged amino acid having a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid having a beta-branched side chain in place of another amino acid having a beta-branched side chain (e.g., He, Thr, and Val), an amino acid having an aromatic side chain in place of another amino acid having, his, Phe, Trp, and Tyr), and the like.
The CAR can consist essentially of one or more specific amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.
The CAR (including functional portions and functional variants) can be of any length, i.e., can comprise any number of amino acids, so long as the CAR (or functional portion or functional variant thereof) retains its biological activity, e.g., the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal, etc. For example, the CAR can be about 50 to about 5000 amino acids long, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.
CARs (including functional portions and functional variants of the invention) may comprise synthetic amino acids substituted for one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino-N-decanoic acid, homoserine, S-acetamidomethyl-cysteine, trans-3-and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β -phenylserine, β -hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid monoamide, N '-benzyl-N' -methyl-lysine, N-acetyl-3-hydroxy-phenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β -phenylserine, β -hydroxyphenylserine, N ', N' -dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, a-aminocyclohexanecarboxylic acid, a-aminocycloheptane-carboxylic acid, a- (2-amino-2-norbornane) -carboxylic acid, gamma-diaminobutyric acid, beta-diaminopropionic acid, homophenylalanine and a-tert-butylglycine.
The CAR (including functional moieties and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (by, for example, disulfide bridges) or converted to an acid addition salt and/or optionally dimerized or multimerized, or conjugated.
The CAR (including functional portions and functional variants thereof) may be obtained by methods known in the art. The CAR can be prepared by any suitable method of preparing a polypeptide or protein. Suitable methods for de novo synthesis of polypeptides and proteins are described, for example, in the following references: chan et al, Fmoc Solid Phase Peptide Synthesis, Oxford university Press, Oxford, United Kingdom, 2000; peptide and Protein drug analysis, ed.reid, r., Marcel Dekker, inc., 2000; epitopic Mapping, ed.westwood et, Oxford University Press, Oxford, United Kingdom, 2001; and us patent 5,449,752. In addition, polypeptides and proteins can be recombinantly produced using nucleic acids described herein using standard recombinant methods. See, e.g., Sambrook et al, Molecular Cloning: a Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al, Current Protocols in molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Additionally, some CARs (including functional portions and functional variants thereof) can be isolated and/or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans), and the like. Methods of isolation and purification are well known in the art. Alternatively, the CARs described herein (including functional portions and functional variants thereof) can be commercially synthesized by a company. In this regard, the CAR can be synthetic, recombinant, isolated, and/or purified.
B. Antibodies and antigen binding fragments
One embodiment also provides a CAR, a T cell expressing a CAR, an antibody, or an antigen binding domain or portion thereof that specifically binds to one or more antigens disclosed herein. As used herein, "CAR-expressing T cell" or "CAR T cell" means a T cell that expresses a CAR and has antigen specificity determined by, for example, the antibody-derived targeting domain of the CAR.
As used herein, "antigen binding domain" may include antibodies and antigen binding fragments thereof. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Some non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for an antigen.
A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. In some examples, a monoclonal antibody is an antibody produced by a monoclonal of B lymphocytes or by cells or progeny thereof into which has been transfected nucleic acids encoding the antibody light and heavy chain variable regions (or antigen-binding fragments thereof) of a single antibody. In some examples, the monoclonal antibody is isolated from the subject. Monoclonal antibodies may have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin function. Exemplary methods for producing monoclonal Antibodies are known, see, e.g., Harlow & Lane, Antibodies, A laboratory Manual, 2nd ed.Cold Spring Harbor Publications, New York (2013).
Generally, immunoglobulins have a heavy (H) chain and a light (L) chain linked to each other by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, and mu constant region genes, as well as myriad immunoglobulin variable domain genes. There are two types of light chains: lambda (. lamda.) and kappa (. kappa.). There are five main heavy chain species (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE.
Each of the heavy and light chains comprises a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al, Kuby Immunology, 6thed., W.H.Freeman and Co., page 91 (2007)). In some embodiments, the heavy and light chain variable regions combine to specifically bind to an antibodyAnd (6) originally. In other embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies consisting of only heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al, Nature, 363: 446. Biol. 448, 1993; Sherff et al, Nat. struct. biol. 3: 733. 736, 1996). Reference to "VH" or "VH" refers to the variable region of an antibody heavy chain, including that of an antigen-binding fragment such as Fv, ScFv, dsFv or Fab. Reference to "VL" or "VL" refers to the variable domain of an antibody light chain, including that of an Fv, ScFv, dsFv, or Fab.
The light and heavy chain variable regions comprise a "framework" region interrupted by three hypervariable regions, also known as "complementarity determining regions" or "CDRs" (see, e.g., Kabat et al, Sequences of Proteins of immunological interest, u.s.department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody (i.e., the combined framework regions that make up the light and heavy chains) are used to locate and align the CDRs in three-dimensional space.
The CDRs are primarily responsible for binding to an epitope of the antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known protocols, including those described by: kabat et al ("sequence of Immunological Interest," 5)thPublic Health Service, national institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et Al, (JMB 273, 927-; "Chothia" numbering scheme) and Lefranc et al ("IMGT unique number for immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains," dev. 55-77, 2003; the "IMGT" numbering scheme). The CDRs of each chain are typically referred to as
An "antigen-binding fragment" is a portion of a full-length antibody that retains the ability to specifically recognize a homologous antigen, as well as various combinations of such portions. Some non-limiting examples of antigen binding fragments include Fv, Fab '-SH, F (ab') 2; a diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments generated by modification of whole antibodies or those synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, vols.1-2, 2nd Ed., Springer Press, 2010).
Single chain antibodies (scFv) are genetically engineered molecules comprising VH and VL domains of one or more antibodies linked by a suitable polypeptide linker to a genetically fused single chain molecule (see, e.g., Bird et al, Science, 242: 423426, 1988; Huston et al, Proc. Natl. Acad. Sci., 85: 58795883, 1988; Ahmad et al, Clin. Dev. Immunol., 2012, doi: 10.1155/2012/980250; Marbry, IDrugs, 13: Acone 549, 2010). The intramolecular orientation of the VH and VL domains in an scFv is generally not critical for scfvs. Thus, scFv having two possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) may be used.
In dsFv, the heavy and light chain variable chains have been mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent, bispecific antibodies: in which the VH and VL domains are expressed on a single polypeptide chain, but a linker is used which is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and creating two antigen binding sites (see, e.g., Holliger et al, Proc. Natl. Acad. Sci., 90: 64446448, 1993; Poljak et al, Structure, 2: 11211123, 1994).
Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-; kuby, j., Immunology, 3rd ed., wh. freeman & co., New York, 1997.
Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly, or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy and variable light chains, such as, for example, Huse et al, Science 246: 1275-1281(1989), which is incorporated herein by reference. These and other methods of making, for example, chimeric, humanized, CDR grafted, single chain and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, immunol. today 14: 243. sup. 246 (1993); Ward et al, Nature 341: 544. sup. 546 (1989); Harlowand Lane, supra, 1988; Hilyard et al, Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2 d. (Oxford university Press 1995); each of which is incorporated herein by reference).
An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and vice versa, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.
A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. A non-human antibody or antigen-binding fragment that provides a CDR is referred to as a "donor", and a human antibody or antigen-binding fragment that provides a framework is referred to as a "recipient". In one embodiment, all CDRs in the humanized immunoglobulin are from a donor immunoglobulin. The constant region need not be present, but if it is present, it can be substantially identical to a human immunoglobulin constant region, e.g., at least about 85% to 90% identical, e.g., about 95% or more. Thus, all parts of the humanized antibody or antigen-binding fragment (except possibly the CDRs) are substantially identical to the corresponding parts of the natural human antibody sequence.
A "chimeric antibody" is an antibody comprising sequences derived from two different antibodies, which are typically of different species. In some examples, a chimeric antibody comprises one or more CDRs and/or framework regions from one human antibody and CDRs and/or framework regions from another human antibody.
A "fully human antibody" or "human antibody" is an antibody that comprises sequences from (or derived from) the human genome and does not comprise sequences from another species. In some embodiments, the human antibody comprises CDRs from (or derived from) a human genome, framework regions, and (if present) an Fc region. Human antibodies can be identified and isolated using techniques for generating antibodies based on sequences derived from the human genome, e.g., by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manual.1st Ed. New York: Cold Spring harbor Laboratory Press, 2004. print.; Lonberg, nat. Biotech., 23: 1117. Snap.1125, 2005; Lonenberg, Curr. Opin. Immunol., 20: 450. J.459, 2008).
An antibody may have one or more binding sites. If more than one binding site is present, the binding sites may be the same as each other or may be different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.
Methods of testing the ability of an antibody to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as Radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al, supra, U.S. patent application publication No.2002/0197266a1 and U.S. patent No.7,338,929).
In addition, the CAR, T cell expressing the CAR, antibody, or antigen-binding portion thereof can be modified to include detectable labels, such as radioisotopes, fluorophores (e.g., Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).
C. Conjugates
A CAR, CAR-expressing T cell, or monoclonal antibody or antigen-binding fragment thereof having specificity for one or more antigens disclosed herein can be conjugated to an agent, e.g., an effector molecule or detectable label, using any number of methods known to those skilled in the art. Both covalent and non-covalent attachment methods may be used. Conjugates include, but are not limited to, molecules in which an effector molecule or detectable label is covalently linked to an antibody or antigen-binding fragment that specifically binds to one or more antigens disclosed herein. One skilled in the art will appreciate that a variety of effector molecules and detectable labels may be used, including, but not limited to, chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents (e.g., as described above)125I、32P、14C、3H and35s) and other labels, target moieties and ligands, etc.
The choice of a particular effector molecule or detectable label depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, the effector molecule may be a cytotoxin that is used to cause death of a particular target cell (e.g., a tumor cell).
The procedure used to attach the effector molecule or detectable label to the antibody or antigen-binding fragment varies depending on the chemical structure of the effector. The polypeptide typically comprises a variety of functional groups, such as carboxylic acid groups (COOH), free amine groups (-NH), which can be used to react with suitable functional groups on the antibody to bind an effector molecule or detectable label2) Or a mercapto group (-SH). Alternatively, the antibody or antigen-binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization may involve the attachment of any of a variety of known linker molecules (e.g., those available from Pierce Chemical Company, Rockford, IL). The linker may be any molecule used to link the antibody or antigen-binding fragment to an effector molecule or detectable label. The linker can be conjugated to the antibody or antigen binding fragment and to the effector molecule or detectable labelWhen the antibody or antigen binding fragment and the effector molecule or detectable label is a polypeptide, the linker may be attached to a constitutive amino acid through its side groups (e.g., through disulfide bonds to cysteine) or to the α carbon amino and carboxyl groups of the terminal amino acids.
In several embodiments, the linker may comprise a spacer element, which when present, increases the size of the linker such that the distance between the effector molecule or detectable label and the antibody or antigen binding fragment increases. Exemplary spacers are known to those of ordinary skill and include those listed in: U.S. patent nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, and U.S. patent publication nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.
In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the effector molecule or detectable label from the antibody or antigen binding fragment in an intracellular environment. In other embodiments, the linker is non-cleavable and the effector molecule or detectable label is released, e.g., by antibody degradation. In some embodiments, the linker can be cleaved by a cleavage agent present in the intracellular environment, e.g., within a lysosome or endosome or pocket (caveolea). The linker may be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease, including but not limited to lysosomal or endosomal proteases. In some embodiments, the peptide linker is at least 2 amino acids long or at least 3 amino acids long. However, the linker may be 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, or 15 amino acids long, e.g., 1 to 2, 1 to 3, 2 to 5,3 to 10, 3 to 15, 1 to 5,1 to 10, 1 to 15 amino acids long. Proteases may include cathepsin B and cathepsin D as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, allowing release of the active drug in the target cell (see, e.g., Dubowchik and Walker, 1999, pharm. For example, a peptide linker that can be cleaved by the thiol-dependent protease cathepsin-B (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker) can be used. Further examples of such linkers are described, for example, in U.S. Pat. No.6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No.6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).
In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at a particular pH value. Generally, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, acid-labile linkers that are hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) may be used. (see, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, pharm. therapeutics 83: 67-123; Neville et al, 1989, biol. chem.264: 14653-14661.) such linkers are relatively stable under neutral pH conditions (e.g., those in blood), but are unstable below pH 5.5 or pH 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether linked to the therapeutic agent via an acylhydrazone bond) (see, e.g., U.S. patent No.5,622,929).
In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using: SATA (N-succinimide-S-acetylthioacetate), SPDP (N-succinimide-3- (2-pyridyldithio) propionate), SPDB (N-succinimide-3- (2-pyridyldithio) butyrate), and SMPT (N-succinimide-oxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene) -, SPDB, and SMPT. (see, e.g., Thorpe et al, 1987, Cancer Res.47: 5924-. See also U.S. patent No.4,880,935).
In other specific embodiments, the linker is a malonate linker (Johnson et al, 1995, Anticancer Res.15: 1387-93), a maleimidobenzoyl linker (Lau et al, 1995, Bioorg-Med-chem.3 (10): 1299-1304), or a 3' -N-amide analog (Lau et al, 1995, Bioorg-Med-chem.3 (10): 1305-12).
In other embodiments, the linker is non-cleavable and the effector molecule or detectable label is released by degradation of the antibody. (see U.S. publication No.2005/0238649, which is incorporated herein by reference in its entirety).
In several embodiments, the linker is resistant to cleavage in an extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., in plasma), no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linker in the conjugate sample is cleaved. Whether a linker is resistant to cleavage in the extracellular environment can be determined, for example, by: the conjugate comprising the linker of interest is incubated with plasma for a predetermined period of time (e.g., 2 hours, 4 hours, 8 hours, 16 hours, or 24 hours), and then the amount of free effector molecule or detectable label present in the plasma is quantified. Various exemplary linkers useful in the conjugates are described in WO 2004-010957, U.S. publication No.2006/0074008, U.S. publication No.20050238649, and U.S. publication No.2006/0024317, each of which is incorporated herein by reference in its entirety.
In several embodiments, conjugates of the CAR, the T cell expressing the CAR, the antibody, or antigen binding portion thereof, and one or more small molecule toxins, such as calicheamicin (calicheamicin), maytansinoids, dolastatins, auristatins, trichothecenes, and CC1065, as well as toxin-active derivatives of these toxins, are provided.
Maytansinoid compounds suitable for use as maytansinoid toxin moieties are well known in the art and may be isolated from natural sources according to known methods, may be produced using genetic engineering techniques (see Yu et al (2002) PNAS 99: 7968-7973), or may be maytansinol and maytansinol analogs prepared synthetically according to known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (Maytenus serrata) (U.S. Pat. No.3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No.4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in the following: U.S. Pat. Nos. 4,137,230; 4,248,870, respectively; 4,256,746, respectively; 4,260,608, respectively; 4,265,814, respectively; 4,294,757, respectively; 4,307,016, respectively; 4,308,268, respectively; 4,308,269, respectively; 4,309,428, respectively; 4,313,946, respectively; 4,315,929, respectively; 4,317,821, respectively; 4,322,348, respectively; 4,331,598, respectively; 4,361,650, respectively; 4,364,866, respectively; 4,424,219, respectively; 4,450,254, respectively; 4,362,663 and 4,371,533, each of which is incorporated herein by reference. Conjugates comprising maytansinoids, methods for their preparation, and their therapeutic use are described, for example, in U.S. Pat. nos. 5,208,020; 5,416,064; 6,441,163 and european
The additional toxin can be used with the CAR, the T cell expressing the CAR, the antibody, or an antigen binding portion thereof. Exemplary toxins include Pseudomonas Exotoxin (PE), ricin, abrin, diphtheria toxin and subunits thereof, ribotoxin (ribotoxin), ribonuclease, saporin, and calicheamicin, and botulinum toxins a through F. These toxins are well known in the art and many are readily available from commercial sources (e.g., Sigma Chemical Company, st. Contemplated toxins also include variants of the toxins (see, e.g., U.S. patent nos. 5,079,163 and 4,689,401).
Saporin is a toxin derived from Saponaria officinalis (Saponaria officinalis) that disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex (stir et al, Bio/Technology, 10: 405-412, 1992). However, this toxin does not have a mechanism for specific entry into the cell, and therefore needs to be conjugated to an internalized antibody or antigen-binding fragment that recognizes a cell surface protein for efficient uptake by the cell.
Diphtheria toxin was isolated from Corynebacterium diphtheriae (Corynebacterium diphtheriae). Typically, diphtheria toxins used in immunotoxins are mutated to reduce or eliminate non-specific toxicity. A mutant called CRM107 with complete enzymatic activity but significantly reduced non-specific toxicity was known since the 70's of the 20 th century (Laird and Groman, J.Virol.19: 220, 1976) and has been used in human clinical trials. See U.S. Pat. No.5,792,458 and U.S. Pat. No.5,208,021.
Ricin is the lectin RCA60 from Castor (Ricinus communis) (Castor bean). For some examples of ricin, see U.S. patent No.5,079,163 and U.S. patent No.4,689,401. Ricinus Communis Agglutinin (RCA) exists in two forms, which are designated RCA according to their molecular weights of about 65kD and 120kD, respectively60And RCA120(Nicholson&Blaustein, j.biochim.biophysis.acta266: 543, 1972). The a chain is responsible for inactivating protein synthesis and killing cells. The B chain binds ricin to cell surface galactose residues and facilitates transport of the A chain into the solute sol (Olsnes et al, Nature 249: 627-631, 1974 and U.S. Pat. No.3,060,165).
Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al, Nat. Biotech.17: 265-70, 1999). Exemplary ribotoxins such as alpha-sarcin and restrictocin are described, for example, in ratore et al, Gene 190: 31-5, 1997; and Goyal and Batra, biochem.345 Pt 2: 247-54, 2000. Calicheamicin was first isolated from Micromonospora echinospora (Micromonospora echinospora) and is a member of the enediyne antitumor antibiotic family causing double strand breaks in DNA that lead to apoptosis (see, e.g., Lee et al, j.Antibiott.42: 1070-87, 1989). This drug is the toxic part of immunotoxins in clinical trials (see, e.g., Gillespie et al, ann. oncol.11: 735-41, 2000).
Abrin includes toxic lectins from Abrus (Abrus precatorius). The toxic components abrin a, B, c and d have molecular weights of about 63kD to 67kD and are composed of two disulfide-linked polypeptide chains a and B. The A chain inhibits protein synthesis; the B chain (abrin-B) binds to a D-galactose residue (see, Funatsu et al, Agr. biol. chem.52: 1095, 1988; and Olsnes, Methods enzymol.50: 330-.
CARs, CAR-expressing T cells, monoclonal antibodies, antigen-binding fragments thereof having specificity for one or more antigens disclosed herein can also be conjugated to a detectable label; for example, a detectable label that is capable of being detected by: ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (e.g., Computed Tomography (CT), Computed Axial Tomography (CAT) scanning, Magnetic Resonance Imaging (MRI), Nuclear Magnetic Resonance Imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optics, and laparoscopy). Specific non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzyme linkers, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers such as luciferase, Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP) are also used. The CAR, T cell expressing the CAR, antibody or antigen binding portion thereof can also be conjugated to an enzyme that can be used for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the CAR, T cell expressing the CAR, antibody or antigen binding portion thereof is conjugated to a detectable enzyme, it can be detected by adding additional reagents used by the enzyme to produce a reaction product that can be discerned. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. The CAR, T cell expressing the CAR, antibody, or antigen binding portion thereof can also be conjugated to biotin and detected by indirectly measuring avidin or streptavidin binding. It should be noted that avidin may itself be conjugated to an enzyme or fluorescent label.
The CAR, the T cell expressing the CAR, the antibody, or antigen binding portion thereof can be conjugated to a paramagnetic agent (e.g., gadolinium). Paramagnetic agents (e.g., superparamagnetic iron oxides) are also used as labels. Antibodies can also be conjugated to lanthanides (e.g., europium and dysprosium) and manganese. The antibody or antigen-binding fragment may also be labeled with a predetermined polypeptide epitope (e.g., a leucine zipper pair sequence, a binding site for a second antibody, a metal binding domain, an epitope tag) that is recognized by a second reporter.
The CAR, T cell expressing the CAR, antibody, or antigen binding portion thereof can also be conjugated to a radiolabeled amino acid. Radiolabels may be used for both diagnostic and therapeutic purposes, for example, radiolabels may be used to detect one or more antigens and antigen-expressing cells disclosed herein by x-ray, emission spectroscopy or other diagnostic techniques. In addition, the radioactive label can be used therapeutically as a toxin for treating a tumor in a subject, e.g., for treating neuroblastoma. Some examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides:3H、14C、15N、35S、90Y、99Tc、111In、125I、131I。
methods for detecting such detectable labels are well known to those skilled in the art. Thus, for example, radioactive labels may be detected using a film or scintillation counter, and fluorescent labels may be detected using a photodetector to detect the emitted illumination. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
D. Nucleotides, expression, vectors and host cells
One embodiment of the invention also provides a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies, or antigen-binding portions thereof (including functional portions and functional variants thereof) described herein. The nucleic acid of the invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen binding domains, transmembrane domains and/or intracellular T cell signaling domains described herein.
In some embodiments, the nucleotide sequence may be codon modified. Without being bound by a particular theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of a nucleotide sequence can involve replacing a native codon with another codon that encodes the same amino acid but that can be translated by a tRNA that is more readily available in the cell, thereby increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that may interfere with translation, thereby increasing translation efficiency.
In one embodiment of the invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding the antigen binding domain of the CAR of the invention. In another embodiment of the invention, the nucleic acid can comprise a codon-modified nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof).
As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally means DNA or RNA polymers, which may be single-stranded or double-stranded, synthetic, or obtained (e.g., isolated and/or purified) from a natural source; it may comprise natural, non-natural or altered nucleotides; and it may comprise natural, non-natural or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, instead of the phosphodiester present between nucleotides of the unmodified oligonucleotide. In some embodiments, the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, as discussed herein, in some cases it may be suitable for a nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
A recombinant nucleic acid can be a nucleic acid having a sequence that does not occur naturally or has a sequence that has been prepared by artificially combining two otherwise isolated segments of sequence. Such artificial combination is typically achieved by chemical synthesis or, more commonly, by artificial manipulation of isolated nucleic acid fragments, e.g., by genetic engineering techniques such as those described in Sambrook et al (supra). Nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, e.g., Sambrook et al (supra) and Ausubel et al (supra). For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to enhance the biological stability of the molecule or to enhance the physical stability of the diad formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Some examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β -D-galactosyltetraoside (queosine), inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β -D-mannosylbousine, 5' -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), wybutoxoside (wybutoxosine), pseudouracil, boudouside, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3- (3-amino-3-N-2-carboxypropyl) uracil and 2, 6-diaminopurine. Alternatively, one or more nucleic acids of the invention may be purchased from companies, such as Integrated DNATechnologies (Coralville, IA, USA).
The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any one of the CARs or a functional portion or functional variant thereof. Alternatively, the nucleotide sequence may comprise a degenerate nucleotide sequence or a combination of degenerate sequences of any one of the sequences.
One embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to or hybridizes under stringent conditions to a nucleotide sequence of any of the nucleic acids described herein.
Nucleotide sequences that hybridize under stringent conditions can hybridize under high stringency conditions. By "high stringency conditions" is meant that a nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions that can distinguish polynucleotides having exactly complementary sequences, or polynucleotides that contain only a few discrete mismatches, from random sequences that occasionally have some small region (e.g., 3 to 10 bases) that matches the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length complements having 14 to 17 bases or more, and high stringency hybridization makes them readily distinguishable. Relatively high stringency conditions can include, for example, low salt and/or high temperature conditions, such as provided by about 0.02M to 0.1M NaCl, or equivalent, at a temperature of about 50 ℃ to 70 ℃. Such high stringency conditions allow for little, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting expression of any of the CARs of the invention. It is generally understood that conditions can be made more stringent by the addition of increasing amounts of formamide.
Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or higher, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein.
In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any one of the nucleic acids. For purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that allows a host cell to express an mRNA, protein, polypeptide, or peptide when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide and the vector is contacted with the cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed within the cell. The carrier as a whole is not naturally occurring.
However, portions of the vector may be naturally occurring. Recombinant expression vectors may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-or double-stranded, synthetic or partially obtained from natural sources, and which may comprise natural, non-natural or altered nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not prevent transcription or replication of the vector.
In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. The vector may be selected from the pUC series (Fermentas Life Sciences, GlenBurnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.).
Phage vectors such as
A variety of transfection techniques are well known in the art (see, e.g., Graham et al, Virology, 52: 456-467 (1973); Sambrook et al (supra); Davis et al, Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al, Gene, 13: 97 (1981)).
Transfection methods include calcium phosphate co-precipitation (see, e.g., Graham et al, supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22: 479-488(1980)), electroporation (see, e.g., Shigekawaet al, BioTechniques, 6: 742-751(1988)), liposome-mediated gene transfer (see, e.g., Manninoet al, BioTechniques, 6: 682-690(1988)), lipid-mediated transduction (see, e.g., Feigner et al, Proc. Natl. Acad. Sci. USA, 84: 7413-7417(1987)), and nucleic acid delivery using high-speed microprojectiles (see, e.g., Klein et al, Nature, 327: 70-73 (1987)).
In one embodiment, recombinant expression vectors can be prepared using standard recombinant DNA techniques such as those described in Sambrook et al (supra) and Ausubel et al (supra). Circular or linear expression vector constructs can be prepared to contain replication systems that are functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, and the like.
Recombinant expression vectors can contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector is to be introduced, as the case may be, and with regard to whether the vector is DNA-based or RNA-based. The recombinant expression vector may contain restriction sites to facilitate cloning.
The recombinant expression vector may comprise one or more marker genes that allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.); complementation to provide prototrophy in an auxotrophic host, and the like. Suitable marker genes for use in the expression vectors of the invention include, for example, the neomycin/G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.
The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding a CAR (including functional portions and functional variants thereof) or to a nucleotide sequence complementary or hybridizing to a nucleotide sequence encoding a CAR. The choice of promoter (e.g., strong, weak, inducible, tissue-specific, and development-specific) is within the ordinary skill of the artisan. Similarly, combinations of nucleotide sequences and promoters are also within the skill of the artisan. The promoter may be a non-viral promoter or a viral promoter, such as the Cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, or a promoter found in the long terminal repeats of murine stem cell viruses.
Recombinant expression vectors can be designed for transient expression, for stable expression, or for both. In addition, recombinant expression vectors can be made for constitutive expression or for inducible expression.
In addition, recombinant expression vectors can be prepared to include suicide genes. The term "suicide gene" as used herein refers to a gene that causes the death of a cell that expresses the suicide gene. The suicide gene may be the following gene: which confers sensitivity to an agent (e.g., a drug) to a cell in which the gene is expressed and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art (see, e.g., suide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), HumanaPress, 2004) and include, e.g., Herpes Simplex Virus (HSV) Thymidine Kinase (TK) Gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.
One embodiment also provides a host cell comprising any of the recombinant expression vectors described herein. The term "host cell" as used herein refers to any type of cell that may comprise a recombinant expression vector of the invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus or algae, or may be a prokaryotic cell, such as a bacterium or protozoa. The host cell may be a cultured cell or a primary cell, i.e. isolated directly from an organism (e.g. a human). The host cell may be an adherent cell or a suspension cell, i.e., a cell grown in suspension. Suitable host cells are known in the art and include, for example, DH5 α escherichia coli (e.coli) cells, chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the purpose of amplifying or replicating the recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5 α cell. For the purpose of producing the recombinant CAR, the host cell can be a mammalian cell. The host cell may be a human cell. While the host cell may be of any cell type, may be derived from any type of tissue, and may be at any developmental stage, the host cell may be a Peripheral Blood Lymphocyte (PBL) or a Peripheral Blood Mononuclear Cell (PBMC). The host cell may be a T cell.
For purposes herein, a T cell may be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, T cells may be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells may also be enriched or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cells may be any type of T cell and may be at any stage of development, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells (i.e., Tscm), naive T cells, and the like. The T cells may be CD8+ T cells or CD4+ T cells.
In one embodiment, the CARs described herein can be used in suitable non-T cells. Such cells are those with immune effector functions, such as NK cells and T-like cells produced by pluripotent stem cells.
One embodiment also provides a cell population comprising at least one host cell described herein. The cell population can be a heterogeneous population comprising host cells containing any one of the recombinant expression vectors and at least one other cell, e.g., a host cell (e.g., a T cell) that does not contain any one of the recombinant expression vectors or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, a red blood cell, a liver cell, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, wherein the population comprises (e.g., consists essentially of) host cells comprising the recombinant expression vector. The population can also be a clonal population of cells, wherein all cells in the population are clones of a single host cell comprising the recombinant expression vector, such that all cells in the population comprise the recombinant expression vector. In one embodiment of the invention, the cell population is a clonal population comprising host cells comprising a recombinant expression vector as described herein.
The CAR (including functional portions and variants thereof), nucleic acid, recombinant expression vector, host cell (including populations thereof), and antibody (including antigen binding portions thereof) can be isolated and/or purified. For example, a purified (or isolated) host cell preparation is one in which the host cell is more pure than the cell in its natural environment in vivo. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, a preparation of host cells is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cellular content of the preparation. For example, the purity can be at least about 50%, can be greater than about 60%, about 70%, or about 80%, or can be about 100%.
E. Method of treatment
The CARs disclosed herein are expected to be useful in methods of treating or preventing disease in a mammal. In this regard, one embodiment provides a method of treating or preventing cancer in a mammal comprising administering to the mammal a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and/or antigen-binding portion thereof, and/or a pharmaceutical composition in an amount effective to treat or prevent cancer in the mammal.
One embodiment further comprises lymphodepletion (lymphodeplate) of the mammal prior to administration of the CAR disclosed herein. Some examples of lymphocyte clearance include, but may not be limited to, non-myeloablative lymphocyte clearance chemotherapy, systemic irradiation, and the like.
For the purposes of the method in which the host cell or population of cells is administered, the cells may be mammalian allogeneic or autologous cells. Preferably, the cells are autologous to the mammal. As used herein, allogeneic refers to any material that is derived from a different animal of the same species as the individual into which the material is introduced. When the genes of one or more loci are not identical, two or more individuals are considered allogeneic to each other. In some aspects, allogeneic material from individuals of the same species may be sufficiently genetically dissimilar to interact antigenically. As used herein, "autologous" means any material that originates from the same individual as the individual into which the material is later reintroduced.
The mammal referred to herein may be any mammal. The term "mammal" as used herein refers to any mammal, including but not limited to mammals of the order rodentia, such as mice and hamsters; and mammals of the order lagomorpha, such as rabbits. The mammal may be from the order carnivora, including felines (felines) and canines (canines). The mammal may be from the order artiodactyla, including bovidae (cattle) and swine (pig); or of the order perssodactyla, including equine (horse). The mammal may be from the order primates, ceboids or simoids (monkeys); or of the order simianidae (human and ape). Preferably, the mammal is a human.
With respect to the method, the cancer may be any cancer, including any of the following: ALL, AML, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulva cancer, Chronic Lymphocytic Leukemia (CLL), chronic myeloid Cancer (CML), colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, NHL, B-chronic lymphocytic leukemia, Hairy cell leukemia, Burkitt's lymphoma, ovarian cancer, pancreatic cancer, cancer of the peritoneum, cancer of the omentum and mesenterium, cancer of the pharynx, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer and cancer of ureter.
The terms "treatment" and "prevention" and words derived therefrom as used herein do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would consider to have potential benefit or therapeutic effect. In this aspect, the method can provide any amount or any level of cancer treatment or prevention in a mammal.
In addition, the treatment or prevention provided by the methods can include treating or preventing one or more conditions or symptoms of a disease being treated or prevented (e.g., cancer). In addition, for purposes herein, "preventing" may encompass delaying the onset of the disease or a symptom or condition thereof.
Another embodiment provides a method of detecting the presence of cancer in a mammal comprising: (a) contacting a sample comprising one or more cells from a mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and/or antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; (b) and detecting the complex, wherein detection of the complex is indicative of the presence of cancer in the mammal.
The sample may be obtained by any suitable method (e.g., biopsy or necropsy). Biopsy is the removal of tissue and/or cells from an individual. Such removal may be for the purpose of collecting tissue and/or cells from an individual for performing an experiment on the removed tissue and/or cells. The experiment may include an experiment to determine whether an individual has and/or is suffering from a particular disorder or disease state. The condition or disease may be, for example, cancer.
For one embodiment of a method of detecting the presence of a proliferative disorder (e.g., cancer) in a mammal, the sample comprising cells of the mammal can be a sample comprising whole cells, whole cell lysates, or fractions of whole cell lysates (e.g., nuclear or cytoplasmic fractions, whole protein fractions, or nucleic acid fractions). If the sample comprises whole cells, the cells may be any cells of the mammal, for example cells of any organ or tissue, including blood cells or endothelial cells.
For mammals, the contacting may occur in vitro or in vivo. Preferably, the contacting is in vitro.
In addition, detection of the complex can be performed by any number of means known in the art. For example, the herein disclosed CARs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies or antigen-binding portions thereof described herein can be labeled with detectable labels, such as radioisotopes, fluorophores (e.g., Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles) as disclosed above.
Methods of testing the CAR's ability to recognize target cells and antigen specificity are known in the art. For example, Clay et, j.immunol, 163: 507-513(1999) teaches methods for measuring the release of cytokines such as interferon- γ, granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor α (TNF- α), or interleukin 2 (IL-2). Additionally, CAR function can be assessed by measuring cellular cytotoxicity, as described by Zhao et al, j. immunol, 174: 4415 as described in 4423 (2005).
Another embodiment provides the use of a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody or antigen-binding portion thereof, and/or a pharmaceutical composition of the invention for treating or preventing a proliferative disorder (e.g., cancer) in a mammal. The cancer may be any one of the cancers described herein.
Any method of administration may be used for the disclosed therapeutic agents, including topical and systemic administration. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration may be used. The particular mode of administration and dosage regimen will be selected by the attending clinician in view of the particular circumstances of the case (e.g., the subject, the disease state involved, and whether the treatment is prophylactic). In cases where more than one agent or composition is administered, one or more routes of administration may be used; for example, the chemotherapeutic agent may be administered orally and the antibody or antigen-binding fragment or conjugate or composition may be administered intravenously. The method of administration includes injection, in which case the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment or composition is provided in a non-toxic pharmaceutically acceptable carrier such as: water, saline, ringer's solution, dextrose solution, 5% human serum albumin, fixed oil (fixed oil), ethyl oleate, or liposomes. In some embodiments, topical administration of the disclosed compounds can be used, for example, by applying an antibody or antigen-binding fragment to an area of tissue from which a tumor has been removed, or an area suspected of being predisposed to tumorigenesis. In some embodiments, sustained intratumoral (or peritumoral) release of a pharmaceutical formulation comprising a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops, or intravitreally to the eye.
The disclosed therapeutic agents can be formulated in unit dosage forms suitable for single administration of precise dosages. In addition, the disclosed therapeutic agents may be administered in a single dose or in a multiple dose regimen. The multiple dose regimen is the following regimen: wherein the primary course of treatment may have more than one individual dose, for example 1 to 10 doses, followed by administration of further doses at subsequent intervals as required to maintain or potentiate the effect of the composition. Treatment may involve a daily dose or multiple daily doses of the compound over a period of days to months or even years. Thus, the dosage regimen will also be determined based at least in part on the particular needs of the subject to be treated and will depend upon the judgment of the administering physician.
A typical dose of antibody or conjugate may be from about 0.01mg/kg to about 30mg/kg, for example from about 0.1mg/kg to about 10 mg/kg.
In some specific examples, a therapeutic composition comprising one or more conjugates, antibodies, compositions, CARs, CAR T cells, or additional agents is administered to a subject based on a multiple daily dosing regimen (e.g., at least 2 consecutive days, 10 consecutive days, etc.), e.g., for a period of weeks, months, or years. In one example, the conjugate, antibody, composition, or additional agent is administered to the subject for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.
In some embodiments, the disclosed methods comprise providing surgery, radiation therapy, and/or chemotherapy to a subject in combination (e.g., sequentially, substantially simultaneously, or simultaneously) with the disclosed antibody, antigen-binding fragment, conjugate, CAR, or CAR-expressing T cell. Such agents and methods of treatment and therapeutic dosages are known to those skilled in the art and can be determined by the skilled clinician. Formulations and dosing regimens for the additional agents can be used according to the manufacturer's instructions or as determined empirically by the skilled artisan. Formulations and dosing regimens for such Chemotherapy are also described in Chemotherapy Service, (1992) ed, m.c. perry, Williams & Wilkins, Baltimore, Md.
In some embodiments, the combination therapy may comprise administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Some non-limiting examples of additional therapeutic agents that may be used in combination therapy include microtubule binding agents, DNA intercalating or crosslinking agents, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene modulators, and angiogenesis inhibitors. These agents (which are administered in therapeutically effective amounts) and treatments may be used alone or in combination. For example, any suitable anti-cancer or anti-angiogenic agent can be administered in combination with a CAR, CAR-T cell, antibody, antigen-binding fragment, or conjugate disclosed herein. Methods and therapeutic dosages of such agents are known to those of skill in the art and can be determined by the skilled clinician.
Additional chemotherapeutic agents include, but are not limited to: alkylating agents, such as nitrogen mustards (e.g., chlorambucil (chlormethine), cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum-containing compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uracil mustard; antimetabolites such as folic acid (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine (tioguanine)), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids such as podophyllum (podophyllum) (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinblastines (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic/antitumor antibiotics, such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampin, hydroxyurea (hydroxyurea), and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab ozogamicin, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers such as aminolevulinic acid, methyl aminolevulinic acid, porfimer sodium, and verteporfin; and other agents, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, acitinib, bexarotene, bevacizumab, bortezomib, celecoxib, dinebin (dilueukin diftitox), erlotinib, estramustine, gefitinib, hydroxyurea (hydoxycarbamide), imatinib, lapatinib, pazopanib, pentostatin, masopropiol, mitotane, pemetrexed, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib, and tretinoin. The choice of such agents and therapeutic dosages are known to those skilled in the art and can be determined by the skilled clinician.
Combination therapy may provide a synergistic effect and prove synergistic, i.e. the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds used alone. When the active ingredients are: (1) co-formulated in a combined unit dose formulation and administered or delivered simultaneously; (2) delivered alternately or concurrently in separate formulations; or (3) by other protocols, a synergistic effect may be obtained. When delivered alternately, a synergistic effect may be obtained when the compounds are administered or delivered sequentially, for example sequentially in separate syringes by different injections. Generally, during alternation, the effective doses of each active ingredient are administered sequentially, i.e. one after the other, whereas in combination therapy the effective doses of two or more active ingredients are administered together.
In one embodiment, an effective amount of an antibody or antigen-binding fragment, or conjugate thereof, that specifically binds to one or more antigens disclosed herein is administered to a subject having a tumor following an anti-cancer treatment. The immune complex is detected after a sufficient amount of time has elapsed to allow the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on the corresponding cancer cell. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to controls taken prior to treatment indicates that the treatment is not effective, whereas a decrease in immune complexes compared to controls taken prior to treatment indicates that the treatment is effective.
F. Biological medicine composition
Provided herein are biopharmaceutical or biologicai compositions (hereinafter "compositions") for gene therapy, immunotherapy, and/or cell therapy comprising one or more of the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells, in a carrier (e.g., a pharmaceutically acceptable carrier) that specifically binds to one or more antigens disclosed herein. The compositions may be prepared in unit dosage form for administration to a subject. The amount and timing of administration is determined by the treating clinician to achieve the desired outcome. The compositions can be formulated for systemic (e.g., intravenous) or topical (e.g., intratumoral) administration. In one example, the disclosed CARs, or T cells expressing CARs, antibodies, antigen-binding fragments, conjugates are formulated for parenteral administration, such as intravenous administration. Compositions comprising the CARs disclosed herein, or T cells, conjugates, antibodies or antigen-binding fragments expressing the CARs, are useful, for example, in the treatment and detection of tumors, such as, but not limited to, neuroblastoma. In some examples, the compositions can be used for the treatment or detection of cancer. Compositions comprising a CAR, or a T cell, conjugate, antibody or antigen-binding fragment expressing a CAR as disclosed herein are also useful, for example, in the detection of pathological angiogenesis.
The composition for administration can comprise a solution of the CAR, or the T cells expressing the CAR, conjugate, antibody, or antigen-binding fragment, dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers can be used, such as buffered saline and the like. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. These compositions may contain pharmaceutically acceptable auxiliary substances as necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, auxiliary agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of CAR, or T cells expressing the CAR, antibody or antigen-binding fragment or conjugate in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the subject. The actual methods of preparing such dosage forms for gene therapy, immunotherapy and/or cell therapy are known or will be apparent to those skilled in the art.
Typical compositions for intravenous administration comprise about 0.01mg/kg to about 30mg/kg of the antibody or antigen-binding fragment or conjugate per subject per day (or a corresponding dose of CAR comprising the antibody or antigen-binding fragment, or a T cell expressing the CAR, conjugate). The actual method for preparing an administrable composition will be known or apparent to those skilled in the art and is described in more detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).
The CAR, or T cell expressing the CAR, antibody, antigen binding fragment or conjugate can be provided in lyophilized form and rehydrated with sterile water prior to administration, but it is also provided in a sterile solution of known concentration. The CAR, or the T cell expressing the CAR, antibody or antigen binding fragment or conjugate solution is then added to an infusion bag containing 0.9% sodium chloride (USP) and in some cases administered at a dose of 0.5 to 15mg/kg body weight. Considerable experience is available in the art in the administration of antibodies or antigen-binding fragments and conjugate drugs; for example since 1997Antibody drugs have been sold in the united states since their approval. The CAR, or the T cell expressing the CAR, the antibody, the antigen binding fragment, and the conjugate thereof, can be administered by slow infusionAdministration is not by intravenous push (push) or bolus (bolus). In one example, a higher loading dose is administered followed by a maintenance dose at a lower level. For example, an initial loading dose of 4mg/kg of antibody or antigen-binding fragment (or a corresponding dose of conjugate comprising antibody or antigen-binding fragment) may be infused over a period of about 90 minutes, followed by a weekly maintenance dose of 2mg/kg over a period of 30 minutes for 4 to 8 weeks if the previous dose is well tolerated.
The controlled release parenteral formulation can be prepared as an implant, an oily injection, or as a particulate system. For a summary of protein delivery systems, see Banga, a.j., Therapeutic Peptides and Proteins: formulation, Processing, and Delivery Systems, technical Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein (e.g., a cytotoxin or drug) as the central core. In the microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres and microcapsules of less than about 1 μm are generally referred to as nanoparticles, nanospheres and nanocapsules, respectively. The diameter of the capillary is about 5 μm so that only nanoparticles are administered intravenously. The microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug delivery systems, J.Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp.219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A.Kydonieus, ed., Marcel Dekker, Inc.New York, NY, pp.315-339, (1992).
The polymers can be used for ion controlled release of the CARs disclosed herein, or T cells, antibodies or antigen binding fragments or conjugate compositions expressing the CARs. A variety of degradable and non-degradable polymeric matrices for controlled drug delivery are known in the art (Langer, Account Chem. Res.26: 537. sup. 542, 1993). For example, block copolymer poloxamer 407 exists as a viscous but mobile liquid at low temperature, but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al, pharm. Res.9: 425-434, 1992; and Pec et al, J.Parent. Sci. Tech.44 (2): 58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al, int. J. pharm.112: 215-. In another aspect, liposomes are used for controlled release and Drug targeting of lipid encapsulated drugs (Betageri et al, Liposome Drug Delivery Systems, Technical publishing Co., Inc., Lancaster, PA (1993)). Many additional systems for controlled delivery of therapeutic proteins are known (see U.S. Pat. No.5,055,303; U.S. Pat. No.5,188,837; U.S. Pat. No.4,235,871; U.S. Pat. No.4,501,728; U.S. Pat. No.4,837,028; U.S. Pat. No.4,957,735; U.S. Pat. No.5,019,369; U.S. Pat. No.5,055,303; U.S. Pat. No.5,514,670; U.S. Pat. No.5,413,797; U.S. Pat. No.5,268,164; U.S. Pat. No.5,004,697; U.S. Pat. No.4,902,505; U.S. Pat. No.5,506,206; U.S. Pat. No.5,271,961; U.S. Pat. No. 483.
G. Medicine box
In one aspect, kits for using the CARs disclosed herein are also provided. For example, the kit is used to treat a tumor in a subject, or to prepare a CAR T cell that expresses one or more CARs disclosed herein. As disclosed herein, a kit will generally comprise a disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cell. More than one of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells can be included in the kit.
The kit may comprise a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container typically contains a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The indicia or package insert indicates that the composition is used to treat a particular condition.
The marker or packaging insert typically further comprises instructions for using the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells, e.g., in methods of treating or preventing tumors or making CAR T cells. The package insert typically contains instructions, typically contained in commercial packaging for the therapeutic product, that contain information regarding the indications, usage, dosage, administration, contraindications and/or warnings associated with the use of such therapeutic products. The illustrative material may be written in electronic form (e.g., a computer diskette or compact disk) or may be visualized (e.g., a video file). The kit may also contain additional components that facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally comprise means for detecting the label (e.g., an enzyme substrate for enzymatic labeling, a filter device for detecting fluorescent labels, a suitable second label (e.g., a second antibody), etc.). The kit may additionally contain buffers and other reagents normally used in the practice of the particular method. Such kits and suitable contents are well known to those skilled in the art.
Examples
The present invention is further illustrated by the following examples, which should not be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may become apparent to those skilled in the art without departing from the spirit of the invention and/or the scope of the appended claims.
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