Combination therapy using cell immunotherapy and an Anti-cancer agent for treating cancer

Abstract

The present disclosure provides combination therapies of a population of modified immune cells and an anti-cancer agent comprising a small molecule (e.g., a chemotherapy drug) or an ADC for treating cancers, especially those resistant to the anti-cancer agent.

Description

COMBINATION THERAPY USING CELL IMMUNOTHERAPY AND AN ANTICANCER AGENT FOR TREATING CANCERSEQUENCE LISTING

[0001] The sequence listing that is contained in the file named “082971-8003W001”, which is 24,114 bytes and was created on June 4, 2025, is filed herewith by electronic submission and is incorporated by reference herein.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority to US provisional patent application no. 63 / 656,113 filed June 05, 2024, the entire disclosure of which is incorporated herein by reference.TECHNICAL FIELD

[0003] The present application generally relates to combination therapies involving cell immunotherapy and an anti-cancer agent (e.g., an anti-cancer agent comprising a small molecule drug or an antibody-drug conjugate) for treating a cancer, especially a cancer resistant or refractory to the anti-cancer agent.BACKGROUND OF THE INVENTION

[0004] Cancer remains a significant global health concern, with diverse types and challenging treatment options. Conventional cancer therapies, such as chemotherapy and targeted cancer therapy (e.g., antibody drug conjugates), may induce cancer resistance or refractoriness to these therapies. Cancer resistance or refractoriness refers to a scenario where a particular cancer fails to respond adequately or becomes resistant to the effects of a specific cancer therapy. This phenomenon poses a significant challenge in cancer treatment, limiting the effectiveness of conventional therapies. Resistance mechanisms can arise from various factors such as genetic alterations, activation of survival pathways, or the presence of cancer stem cells.

[0005] Therefore, there is a need for developing novel anti-cancer therapies for treating cancers, especially those resistant or refractory to an anti-cancer agent (e.g., an anti-cancer agent comprising a small molecule drug or an antibody-drug conjugate).SUMMARY OF THE INVENTION

[0006] An objective of the present application is to provide a method for treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of modified immune cells in combination with an anti-cancer agent, wherein the subject comprises a cancer cell expressing a cancer surface marker, wherein the modified immune cell comprises a polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell, wherein the CAR comprises (1) an extracellular antigen-binding domain specific for the cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR, and wherein the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

[0007] In another aspect, the present disclosure provides a kit comprising (a) a first composition comprising a population of modified immune cells, and (b) a second composition comprising an anti-cancer agent, wherein the modified immune cell comprises a polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell, wherein the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR, and wherein the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

[0008] In another aspect, the present disclosure provides a kit comprising (a) a first composition comprising a polynucleotide encoding a chimeric antigen receptor (CAR), and (b) a second composition comprising an anti-cancer agent, wherein the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR, and wherein the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

[0009] In another aspect, the present disclosure provides a kit comprising a pharmaceutical composition comprising a population of modified immune cells, and an insert comprising instructions of administering the pharmaceutical composition in combination with an anti-cancer agent, wherein the modified immune cell comprises a polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell, wherein the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR, and wherein the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

[0010] In another aspect, the present disclosure provides a kit comprising a polynucleotide encoding a chimeric antigen receptor (CAR), and an insert comprising instructions of administering an effective amount of modified immune cells comprising the polynucleotide in combination with an anti-cancer agent, wherein the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR, and wherein the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

[0011] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention. Further, the accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain principles of the invention.BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG. 1 illustrates the structure of the chimeric antigen receptor polypeptide.

[0013] FIG. 2 illustrates the tumor growth curve of glioma cell U87 in humanized mice treated with modified DCs in combination with the chemotherapy drug cis-platinum.

[0014] FIG. 3 illustrates the tumor growth curve of colorectal cancer cell HCT116 in humanized mice treated with modified DCs in combination with chemotherapy drug Etoposide.

[0015] FIG. 4 illustrates the tumor growth curve of liver cancer cell HepG2 in humanized mice treated with modified DCs in combination with chemotherapy drug Etoposide.

[0016] FIG. 5 illustrates the tumor growth curve of breast cancer cell MCF7 in humanized mice treated with modified DCs in combination with the chemotherapy drug Doxorubicin.

[0017] FIG. 6 illustrates the tumor growth curve of lung cancer cell line A549 in humanized mice treated with modified DCs in combination with chemotherapy drug paclitaxel.

[0018] FIG. 7 illustrates the tumor growth curve of liver cancer cell line Huh7 in humanized mice treated with modified DCs in combination with chemotherapy drug paclitaxel.

[0019] FIG. 8 illustrates the tumor growth curve of ovarian cancer cell line SKOV3 in humanized mice treated with modified DCs in combination with ADC drug T-DXd.

[0020] FIG.9 illustrates the levels of cytokines after treatment with CARDC and ADC.

[0021] FIG. 10 illustrates the tumor growth curve of breast cancer cell line SKBR3 in humanized mice treated with modified DCs in combination with ADC drug T-DXd.

[0022] FIG.11 illustrates the tumor growth curve of lung cancer cell line A549 in humanized mice treated with modified DCs in combination with ADC drug T-DXd.

[0023] FIG.12 illustrates the tumor growth curve of pancreatic cancer cell line Bxcp3 in humanized mice treated with modified DCs in combination with ADC drug Sacituzumab govitecan.DETAILED DESCRIPTION OF THE INVENTION

[0024] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosurebelongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0026] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0027] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0028] I. General Definitions

[0029] The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over the definition of the term as generally understood in the art.

[0030] The singular terms “a”, “an”, and “the” include plural referents unless context clearly indicates otherwise. By way of example, reference to “a cell” refers to one or more cells, and reference to “the method” includes reference to equivalent steps and methods disclosed herein and / or known to those skilled in the art, and so forth. Similarly, the word "or" is intended to include “and” unless the context clearly indicates otherwise. Although methods and materialssimilar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0031] As used herein, the term “about” or “approximately” means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0032] As used herein, the terms such as “comprises”, “comprised”, “comprising”, “contains”, “containing” and the like have the meaning attributed in United States Patent law; they are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. Terms such as “consisting essentially of’ and “consists essentially of’ have the meaning attributed in United States Patent law; they allow for the inclusion of additional ingredients or steps that do not materially affect the basic and novel characteristics of the claimed invention. The terms “consists of’ and “consisting of’ have the meaning ascribed to them in United States Patent law; namely that these terms are close ended.

[0033] In all occurrences in this application where there are a series of recited numerical values, it is to be understood that any of the recited numerical values may be the upper limit or lower limit of a numerical range. It is to be further understood that the invention encompasses all such numerical ranges, i.e., a range having a combination of an upper numerical limit and a lower numerical limit, wherein the numerical value for each of the upper limit and the lower limit can be any numerical value recited herein. Ranges provided herein are understood to include all values within the range. For example, 1-10 is understood to include all of the values 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional values as appropriate. Similarly, ranges delimited by “at least” are understood to include the lower value provided and all higher numbers.

[0034] As used herein, the term “CAR”, which can be used interchangeably with the term “chimeric antigen receptor” refers to an engineered receptor or a synthetic receptor or polynucleotide encoding thereof. The engineered receptor or a synthetic receptor comprises an extracellular domain that comprises an antigen binding domain, a transmembrane domain, and / or an intracellular signaling domain, optionally a signal peptide, which are joined one another or operably linked to each other. The most common CARs are, for example, a single chain variable fragment (scFv) derived from a monoclonal antibody fused to CD3-zeta transmembrane and endodomain. Such CARs result in the transmission of a zeta signal in response to specific binding of scFv to its target. Methods of preparing CARs are publicly available (see, e.g., Grupp et al., N Engl J Med., 368: 1509-1518, 2013; Park et al., Trends Biotechnol., 29:550-557, 2011; Haso etal.,(2013) Blood, 121, 1165-1174; Han et al., J. Hematol Oncol.6: 47, 2013; WO2012 / 079000; U.S. Pub.2012 / 0213783; and WO2013 / 059593, each of which is incorporated by reference herein in its entirety).

[0035] As used herein, the term “operably linked” refers to a functional relationship between two or more polynucleotide sequences. In the context of a polynucleotide encoding a fusion protein, such as a polypeptide chain of a CAR of the disclosure, the term means that the two or more polynucleotide sequences are joined such that the amino acid sequences encoded by these segments remain in-frame. In the context of transcriptional or translational regulation, the term refers to the functional relationship of a regulatory sequence to a coding sequence, for example, a promoter in the correct location and orientation to the coding sequence so as to modulate the transcription.

[0036] As used herein, the term “Antigen-binding domain” refers to an antibody fragment formed from a portion of an intact antibody comprising one or more CDRs, or any other antibody fragment that can bind to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding domain include, without limitation, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv1), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), single-chain Fv-Fc antibody (scFv-Fc), an scFv dimer (bivalent diabody), a bispecific antibody, a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding domain is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding fragment / portion may comprise one or more CDRs from a particular parent antibody.

[0037] ‘ ‘Fab” with regard to an antibody refers to a monovalent antigen-binding fragment of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond. Fab can be obtained by papain digestion of an antibody at the residues proximal to the N-terminus of the disulfide bond between the heavy chains of the hinge region.

[0038] ‘ ‘Fab'” refers to a Fab fragment that includes a portion of the hinge region, which can be obtained by pepsin digestion of an antibody at the residues proximal to the C-terminus of the disulfide bond between the heavy chains of the hinge region and thus is different from Fab in a small number of residues (including one or more cysteines) in the hinge region.

[0039] “F(ab')2” refers to a dimer of Fab’ that comprises two light chains and part of two heavy chains.

[0040] “ Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site. A Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. A “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0041] “Single-chain Fv antibody” or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879(1988)). A “scFv dimer” refers to a single chain comprising two heavy chain variable regions and two light chain variable regions with a linker. In certain embodiments, an “scFv dimer” is a bivalent diabody or bivalent ScFv (BsFv) comprising VH-VL (linked by a peptide linker) dimerized with another VH- VL moiety such that VH's of one moiety coordinate with the VL's of the other moiety and form two binding sites which can target the same antigens (or epitopes) or different antigens (or epitopes). In other embodiments, a “scFv dimer” is a bispecific diabody comprising VH1-VL2 (linked by a peptide linker) associated with VL1-VH2 (also linked by a peptide linker) such that VH1 and VL1 coordinate and VH2 and VL2 coordinate and each coordinated pair has a different antigen specificity.

[0042] “Single-chain Fv-Fc antibody” or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.

[0043] “ Camelized single domain antibody,” “heavy chain antibody,” “nanobody” or “HCAb” refers to an antibody that contains two VH domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(l-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally obtained from Camelidae (camels, dromedaries, and llamas). Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. Apr;54(l):39-47 (2002); Nguyen VK. et al. Immunology. May;109(l):93-101 (2003)). Thevariable domain of a heavy chain antibody (VHH domain) represents the smallest known antigenbinding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)). “Diabodies” include small antibody fragments with two antigen-binding sites, wherein the fragments comprise a VH domain connected to a VL domain in a single polypeptide chain (VH-VL or VL-VH) (see, e.g., Holliger P. et al., Proc Natl Acad Sci U S A. Jul 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). The two domains on the same chain cannot be paired, because the linker is too short, thus, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same of different antigens (or epitopes).

[0044] A “domain antibody” refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In certain embodiments, two or more VH domains are covalently joined with a peptide linker to form a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.

[0045] In certain embodiments, a “(dsFv)2” comprises three peptide chains: two VH moieties linked by a peptide linker and bound by disulfide bridges to two VL moieties.

[0046] In certain embodiments, a “bispecific ds diabody” comprises VH1-VL2 (linked by a peptide linker) bound to VL1-VH2 (also linked by a peptide linker) via a disulfide bridge between VH1 and VLl.

[0047] In certain embodiments, a “bispecific dsFv” or “dsFv-dsFv1” comprises three peptide chains: a VH1-VH2 moiety wherein the heavy chains are bound by a peptide linker (e.g., a long flexible linker) and paired via disulfide bridges to VL1 and VL2 moieties, respectively. Each disulfide paired heavy and light chain has a different antigen specificity.

[0048] As used herein, the term “cell” as used herein refers to individual cells, cell lines, or cultures derived from such cells.

[0049] As used herein, the term “polynucleotide” or “nucleic acid” refers to a chain of nucleotides. They also refer to synthetic and / or non-naturally occurring nucleic acid molecules (e.g., comprising nucleotide analogues or modified backbone residues or linkages). The terms also refer to deoxyribonucleotide or ribonucleotide oligonucleotides in either single-stranded or doublestranded form. The terms encompass nucleic acids containing analogues of natural nucleotides. The terms also encompass nucleic acid-like structures with synthetic backbones. Unless otherwiseindicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

[0050] As used herein, the term “single-chain variable fragment” used interchangeably with the term “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (Huston JS etal. Proc Natl Acad Sci USA, 85:5879(1988)).

[0051] As used herein, the term “treatment,” “treat” or “treating”, with regard to a disorder, refers to managing, eliminating, reducing or ameliorating a disorder and / or a symptom associated therewith. Although not excluded, treatment of a disorder does not require that the disorder, or symptoms associated therewith be completely eliminated. The term “treatment” as used herein may include “prophylactic treatment” that is applied before development of any symptom or manifestation of a disorder to reduce the possibility of or block the occurrence or recurrence of a disorder, or reducing the possibility of relapse of a previously controlled disorder, in a subject who is not afflicted with a disorder but at risk, or who is susceptible to recurrence of the disorder, or who is at risk or susceptible to relapse of the disorder. Within the meaning of the invention, “treatment” also includes prevention of relapse or prevention stages, as well as treatment of acute or chronic signs, symptoms and / or dysfunction. Treatment can target symptoms, for example, to suppress symptoms. It can function in a short period of time, for a medium period of time, or can be a long-term treatment, such as in the case of maintenance therapy.

[0052] As used herein, administration of one agent (e.g., a population of modified immune cells) “in combination with” another one or more further agents includes simultaneous (concurrent) and consecutive administration in any order. It is to be understood, for example, that Drug A is administered “in combination with” Drug B, or use of Drug A “in combination with” Drug B can encompass at least three scenarios, for example: 1) Drug A and Drug B are formulated in a single dosage form prior to administration or at the time of administration; 2) Drug A and Drug B are formulated in two separate dosage forms and provided (e.g., manufactured or sold) together withinstructions of administering Drug A in combination with Drug B; and 3) Drug A and Drug B are formulated in two separate dosage forms and provided (e.g., manufactured or sold) separately with instructions of either i) administering Drug A in combination with Drug B when Drug A is being provided (e.g., manufactured or sold), or ii) administering Drug B in combination with Drug A when Drug B is being provided (e.g., manufactured or sold). In other words, use of Drug A in combination with Drug B does not necessarily mean that Drug A and Drug B must be provided (e.g., manufactured or sold) together.

[0053] As used herein, the term “effective amount” refers broadly to the amount of a compound or cells that, when administered to a patient for treating a disease, is sufficient to affect such treatment for the disease. The effective amount may be an amount effective for prophylaxis, and / or an amount effective for prevention. The effective amount may be an amount effective to reduce, an amount effective to prevent the incidence of signs / symptoms, to reduce the severity of the incidence of signs / symptoms, to eliminate the incidence of signs / symptoms, to slow the development of the incidence of signs / symptoms, to prevent the development of the incidence of signs / symptoms, and / or effect prophylaxis of the incidence of signs / symptoms. The “effective amount” may vary depending on the disease and its severity and the age, weight, medical history, susceptibility, and preexisting conditions, of the patient to be treated. The term “effective amount” is synonymous with “therapeutically effective amount” for purposes of this invention.

[0054] As used herein, the term “subject” is not limited to a specific species or sample type. For example, the term “subject” may refer to a patient, and frequently a human patient. However, this term is not limited to humans and thus encompasses a variety of mammalian species, such as nonhuman veterinarian mammal, such as dogs, cats, rabbits, pigs, rodents, horses, or monkeys.

[0055] “ Percent (%) sequence identity” or “Percent (%) identity” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum correspondence. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI), see also, Altschul S.F. et al, J. Mol. Biol., 215:403-410 (1990); Stephen F.et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D.G. et al, Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al, Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm. In certain embodiments, the non-identical residue positions may differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference.

[0056] The term “conservative substitution”, as used herein with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Vai, Leu, and He), among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gin), among residues with acidic side chains (e.g. Asp, Glu), among amino acids with basic side chains (e.g. His, Lys, and Arg), or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe). As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.

[0057] The term “functional forms” as used herein, refers to different forms (such as variants, fragments, fusions, derivatives and mimetics) of the parent molecule, which, despite of having difference in amino acid sequences or in chemical structures, still retains substantial biological activity of the parent molecule. The expression “retain substantial biological activity”, as used herein, means exhibiting at least part of (for example, no less than about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or all of the biological activity of the parent molecule. A functional form of a parent polypeptide may include both naturally-occurring variant forms and non-naturallyoccurring forms such as those obtained by recombinant methods or chemical synthesis. The functional forms may contain non-natural amino acid residues.

[0058] The term “vector” as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating. A vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the fusion polypeptide, at least one promoter (e.g., SV40, CMV, EF-la) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40), lambda phage, and Ml 3 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD- Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, p VITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, pl5TV-L, pProl8, pTD, pRSlO, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNAl.l / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXTl, pCDEF3, pSVSPORT, pEF-Bos etc.

[0059] IL Methods of Treating Cancers

[0060] The present disclosure provides a method for treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of modified immune cells incombination with an anti-cancer agent. In certain embodiments, the subject comprises a cancer cell expressing a cancer surface marker. The term “cancer surface marker” as used herein refers to a molecule differentially expressed on surface of a cancer cell to be used as a target for recognizing a cancer cell.

[0061] “ Cancer” as used herein refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration, or metastasis, and includes both solid tumors and non-solid cancers (e.g., hematologic malignancies). As used herein “solid tumor” refers to a solid mass of neoplastic and / or malignant cells.

[0062] In certain embodiments, the cancer is resistant or refractory to the anti-cancer agent. Drug resistance continues to be the principal limiting factor to achieving cures in patients with cancer. The problem of resistance to an anti-cancer agent in cancer is a complex and multifaceted issue. Drug resistance in cancer arises from numerous reasons, such as the burden and growth kinetics of the tumor, tumor heterogeneity, physical barriers posed by the tumor itself, the interplay between the immune system and the microenvironment, the existence of undruggable cancer drivers, and the numerous consequences that arise from the application of therapeutic pressures. See, e.g., Vasan, N., Baselga, J. & Hyman, D.M. A view on drug resistance in cancer. Nature 575, 299-309 (2019). Given this wide array of factors, it becomes increasingly difficult to predict whether a particular therapy will be effective against cancers that have developed drug resistance.

[0063] In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a solid cancer selected from the group consisting of glioma (e.g., glioblastoma), colorectal cancer (e.g., colon cancer), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma (e.g., well- differentiated hepatocellular carcinoma)), breast cancer, and lung cancer (e.g., non-small cell lung cancer) , prostate cancer, pancreatic cancer, ovarian cancer, stomach cancer, esophageal cancer and melanoma.

[0064] In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a hematologic malignancy selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma (MM).

[0065] 1. Modified Immune Cell

[0066] In certain embodiments, the modified immune cell comprises a polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell (DC). The modified immune cell provided herein may comprise or express one or more (for example, 1, 2, 3, or more) CARs. The one or more CARs may be the same or different. In certain embodiments, the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR.

[0067] The activation of a DC can be assayed by measuring various parameters, including, without limitation, the activation status of DC and / or the activation status of immune cells (e.g., T cells, microphages), which can be indicated by the expression level of DC activating markers (such as CD80, CD86 and MHC-II, CD83, CD54, CMRF-44, CMRF-56, type III INF, IL-12, CXCL9 / 10, IRF8), the survival and / or cytotoxicity of the immune cells (e.g., T cells), the expression (and / or secretion) of immune stimulating cytokines (e.g., TNF-a, IFN-a, IFN-P, IFN-y, IL-1, IL-2, IL-4, IL-6, IL-8, IL- 10, IL- 12, IL- 18 and granulocyte-macrophage colony stimulating factor) from the immune cells (e.g., T cells), the expression level of immune inhibitory molecules (e.g., PD-1, TIM- 3, TIGIT, LAG3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, KIR, N0X2, VISTA, SIGLEC7 (CD328), PVR(CD155), and SIGLEC9 (CD329)) from the immune cells (e.g., T cells), and / or the expression level of markers for anti-inflammatory macrophages (e.g., M2 macrophages), such as CD206 and CD 163.

[0068] In certain embodiments, the activation of dendritic cells comprises increased expression level of DC activating markers (such as CD80, CD86 and / or MHC-II, CD83, CD54, CMRF-44, CMRF-56, type III INF, IL-12, CXCL9 / 10, IRF8), increased survival of the immune cells (e.g., T cells (such as CD8+ T cells), DCs), increased expression (and / or secretion) of immune stimulating cytokines (e.g., TNF-a, IFN-a, IFN-P, IFN-y, IL-1, IL-2, IL-4, IL- 6, IL-8, IL-10, IL-12, IL-18 and / or granulocyte-macrophage colony stimulating factor) from the immune cells (e.g., T cells), decreased expression of immune inhibitory molecules (e.g., PD-1, TIM-3, TIGIT, LAG3, A2AR, BTLA (CD272), CTLA-4 (CD 152), IDO1, IDO2, TDO, KIR, N0X2, VISTA, SIGLEC7 (CD328), PVR(CD155), and SIGLEC9 (CD329)) from the immune cells (e.g., T cells), and / or decreased expression level of markers (such as CD206 and CD 163) for anti-inflammatory macrophages (e.g., M2 macrophages), when compared to a reference status (e.g., inactivated status) of dendritic cells.

[0069] (1) Extracellular Antigen-Binding Domain

[0070] In some embodiments, the antigen binding domain comprises a human or humanized antibody or an antibody fragment thereof. The term “human antibody” refers to an antibody where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin. The term “humanized antibody” refers to an antibody which contains sequence (e.g., CDR sequences) derived from non-human immunoglobulin. Human or humanized antibodies or fragments thereof may be prepared in a variety of ways, for example through recombinant methodologies or through immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and / or light chain encoding genes.

[0071] In some embodiments, the extracellular antigen-binding domain of the CAR provided herein comprises a single-chain variable fragment (scFv), a Fv, a Fab, a (Fab)2, an scFv, a nanobody, a non- covalently or covalently linked ligand / receptor domain or any alternative scaffold known in the art to function as antigen binding domain.

[0072] In some embodiments, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv). The scFv can be specific to a cancer surface marker. In certain embodiments, the cancer surface marker is selected from the group consisting of: EphA2, CD 19, CD70, CD117, CD133, CD147, CD171, DLL3, EGFRvin, VGFR2, Mesothelin, ganglioside GD2, FAP (fibroblast activating protein), FBP (folate binding protein), LMP1, Lewis Y, Claudin 18.2, IL13Ra2, HER2, MDC1, PMSA (prostate membrane specific antigen), R0R1, ROR2, B7-H3, CAIX, CD133, CD171, CEA, GPC3, MUC1, MUC16, MAGE-A1, MAGE-A4, TROP2, EpCAM, CD20, BCMA, Trop2 and NKG2D, other proteins found to be more highly enriched on the surface of tumor cells than critical normal tissues, and combination thereof.

[0073] In some embodiments, the scFv is specific for EphA2. The EphA2, a member of the Eph receptor family, exhibits a distinct expression pattern in tumor tissues. Notably, it is overexpressed in tumorous areas, whereas its presence is comparatively scant in most normal adult tissues. This distinctive behavior suggests that EphA2 may hold significant potential for utilization in cancer treatment strategies. See, e.g., Xiao T et al., Targeting EphA2 in cancer. J Hematol Oncol. 2020 Aug 18;13(1):114. In some embodiments, the scFv comprises a variable heavy (VH) and variable light (VL) region. In some embodiments, the VH comprises a heavy chain CDR1 (HCDR1) having a sequence set forth in SEQ ID NO: 8, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof; a CDR2 having a sequence set forth in SEQ ID NO:9, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof; and a CDR3 having a sequence set forth in SEQ ID NO: 10, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof. In some embodiments, the VL region comprises a light chain CDR1 (LCDR1) having a sequence set forth in SEQ ID NO: 11 , or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof; a CDR2 having a sequence set forth in SEQ ID NO: 12, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof; and a CDR3 having a sequence set forth in SEQ ID NO: 13 , or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof.

[0074] In some embodiments, the scFv is specific for trophoblast cell-surface antigen 2 (TROP2). TROP2 is a transmembrane glycoprotein and a calcium (Ca2 +) signal transducer. It is overexpressed in various types of cancer, including breast, lung, gastric, colorectal, pancreatic, prostate, cervical, head and neck cancers, as well as nasopharyngeal and ovarian carcinomas. TROP2 can interact with P-catenin within the Wnt signaling pathway, thereby contributing to the transcription of nuclear oncogenes and promoting cell proliferation. In some embodiments, the scFv comprises a variable heavy (VH) and variable light (VL) region. In some embodiments, the VH comprises a heavy chain CDR1 (HCDR1) having a sequence set forth in SEQ ID NO: 18, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof; a CDR2 having a sequence set forth in SEQ ID NO: 19, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9Y1or 10 conservative substitutions thereof, or any functional forms thereof; and a CDR3 having a sequence set forth in SEQ ID NO: 20, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof. In some embodiments, the VL region comprises a light chain CDR1 (LCDR1) having a sequence set forth in SEQ ID NO: 21, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof; a CDR2 having a sequence set forth in SEQ ID NO: 22, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof; and a CDR3 having a sequence set forth in SEQ ID NO: 23 , or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof.

[0075] In certain embodiments, the scFv comprises 1) a VH comprising a HCDR1 comprising a sequence set forth in SEQ ID NO: 8 , a HCDR2 comprising a sequence set forth in SEQ ID NO: 9 , a HCDR3 comprising a sequence set forth in SEQ ID NO: 10; and 2) a VL comprising a LCDR1 comprising a sequence set forth in SEQ ID NO: 11 , a LCDR2 comprising a sequence set forth in SEQ ID NO: 12, a LCDR3 comprising a sequence set forth in SEQ ID NO: 13. In certain embodiments, the scFv comprises 1) a VH comprising a HCDR1 comprising a sequence set forth in SEQ ID NO: 18 , a HCDR2 comprising a sequence set forth in SEQ ID NO: 19 , a HCDR3 comprising a sequence set forth in SEQ ID NO: 20; and 2) a VL comprising a LCDR1 comprising a sequence set forth in SEQ ID NO: 21 , a LCDR2 comprising a sequence set forth in SEQ ID NO: 22, a LCDR3 comprising a sequence set forth in SEQ ID NO: 23.

[0076] In some embodiments, the scFv comprises a VH and a VL. In certain embodiments, the VH comprises an amino acid sequence set forth in SEQ ID NO: 14, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof. In certain embodiments, the VH comprises an amino acid sequence set forth in SEQ ID NO: 24, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identitythereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof. In certain embodiments, the VL comprises an amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof. In certain embodiments, the VL comprises an amino acid sequence set forth in SEQ ID NO: 25, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof. In some embodiments, the scFv comprises a VH comprising a sequence set forth in SEQ ID NO: 14, and a VL comprising a sequence set forth in SEQ ID NO: 15. In some embodiments, the scFv comprises a VH comprising a sequence set forth in SEQ ID NO: 24, and a VL comprising a sequence set forth in SEQ ID NO: 25.

[0077] In certain embodiments, the scFv comprises a peptide linker of at least 0, 1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In certain embodiments, the peptide linker comprises an amino acid sequence comprising SEQ ID NO: 17 (GGGGSGGGGSGGGGS).

[0078] In some embodiments, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 26.

[0079] The skilled person in the art will appreciate that an appropriate extracellular antigenbinding domain specific for any disease marker may be selected to construct a CAR provided herein, depending on the disease of interest, in view of the existing knowledge of the identified markers for various diseases, such as cancer, infectious diseases, or immune diseases. The various disease markers include but not limited to those as described above.

[0080] (2) Transmembrane Domain

[0081] The transmembrane domain of the CAR described herein may be derived from any membrane-bound or transmembrane protein including, but are not limited to, BAFFR, BLAME (SLAMF8), CD2, CD3 epsilon, CD4, CD5, CD8, CD9, CDlla (CD18, ITGAL, LFA-1), CDllb, CDllc, CDlld, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a,CD49d, CD49f, CD64, CD80, CD84, CD86, CD96 (Tactile), CD 100 (SEMA4D), CD103, CD134, CD137 (4-1BB), CD150 (IPO-3, SLAMF1, SLAM), CD154, CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (Ly9), CD244 (2B4, SLAMF4), CD278 (ICOS), CEACAM1, CRT AM, GITR, HYEM (LIGHTR), IA4, IL2R beta, IL2R gamma, IL7R a, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, 0X40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Lyl08), SLAMF7, an alpha, beta or zeta chain of a T-cell receptor, TNFR2, VLA1, and VLA-6.

[0082] In some embodiments, the transmembrane domain comprises a transmembrane domain of CD8 alpha. In certain embodiments, the transmembrane domain of CD8 alpha has a sequence of SEQ ID NO: 6, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof.

[0083] In certain embodiments, the transmembrane domain of the CAR described herein is synthetic, e.g., comprising predominantly hydrophobic residues such as leucine and valine. In certain embodiment, the transmembrane domain of the CAR described herein is modified or designed to avoid binding to the transmembrane domains of the same or different surface membrane proteins in order to minimize interactions with other members of the receptor complex.

[0084] In some embodiments, the CAR described herein further comprises a hinge region, which forms the linkage between the extracellular domain and transmembrane domain of the CAR. The hinge and / or transmembrane domain provides cell surface presentation of the extracellular antigenbinding domain of the CAR.

[0085] The hinge region may be derived from any membrane-bound or transmembrane protein including, but are not limited to, BAFFR, BLAME (SLAMF8), CD2, CD3 epsilon, CD4, CD5, CD8, CD9, CDlla (CD18, ITGAL, LFA-1), CD 11b, CD 11c, CD lid, CD 16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, CD64, CD80, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134, CD137 (4- IBB), CD150 (IPO-3, SLAMF1, SLAM), CD154, CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (Ly9), CD244 (2B4, SLAMF4), CD278 (ICOS), CEACAM1, CRT AM, GITR, HYEM (LIGHTR), IA4, IL2R beta, IL2R gamma, IL7Ra, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, 0X40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, LylO8), SLAMF7, an alpha, beta or zeta chain of a T-cell receptor, TNFR2, VLA1, and VLA-6.

[0086] In some embodiments, the hinge region comprises a hinge region of CD 8 alpha, a hinge region of human immunoglobulin (Ig), or a glycine-serine rich sequence. In some embodiments, the CAR comprises a hinge region of CD8 alpha. In certain embodiments, the hinge region of CD8 alpha comprises a sequence set forth in SEQ ID NO: 7, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof.

[0087] (3) Intracellular Signaling Domain

[0088] The intracellular signaling domain of the CAR described herein is responsible for activation of at least one of the normal effector functions of the immune cell (e.g., dendritic cell) in which the CAR has been placed in. The term “effector function” used in the context of an immune cell refers to a specialized function of the cell, for example, the phagocytic activity, cytolytic activity or helper activity. In certain embodiments, the intracellular signaling domain of the CAR described herein is capable of activating (including maturation) dendritic cells in an immune suppressive tumor microenvironment. Activation of DCs can be induced by many cell surface receptors, such as TLR4 (A. Iwasaki et al., Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5, 987-995 (2004).), TNFR (L. M. Sedger et al., From mediators of cell death and inflammation to therapeutic giants - past, present and future. Cytokine Growth Factor Rev. 25, 453-472 (2014).), IFNyR (M. Z. Jianping Pan et al., Interferon-y is an autocrine mediator for dendritic cell maturation. Immunol. Lett. 94, 141-151 (2004).), Dectin-1 (T. S. Helen S. et al., Differential utilization of CARD9 by Dectin-1 in macrophages and dendritic cells. J Immunol. 182, 11461154 (2009)) and FcyR (M. Guilliams etal., The function of Fey receptors in dendritic cells and macrophages. Nat. Rev. Immunol. 14, 94-108 (2014)., T. H. Flinsenberg, Fc receptor antigen targeting potentiates cross-presentation by human blood and lymphoid tissue BDCA-3 dendritic cells. Blood 120, 26 (2012).) in response to various stimuli. These DC activating receptors have one or more immune-receptor tyrosine-based activation motif (ITAM) in their cytoplasmic domains, which triggers activating signal cascades to activate DCs. As used herein, the term “cytoplasmic domain” refers to a fully length domain of a protein residing inside cytoplasm, or any fragment thereof, for example, a fragment having a length that is at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the full- length domain.

[0089] The intracellular signaling domain of the CAR described herein may comprise a cytoplasmic domain of a dendritic cell activating receptor selected from the group consisting of RIG-1, NLRP10, DEC-205, BDCA-2, CD86, 4-1BBL, OX40L, CD40, IFNAR, TLR4, TNFR(e.g., TNFR2), IFNyR, Dectin-1 and FcyR, or a combination thereof. In certain embodiments, the intracellular signaling domain of the CAR described herein comprises the cytoplasmic domain of Dectin-1 and the cytoplasmic domain of FcyR.

[0090] In certain embodiments, the cytoplasmic domain of Dectin-1 and the cytoplasmic domain of FcyR are connected in tandem. In certain embodiments, the polynucleotide encoding the cytoplasmic domain of Dectin-1 is upstream the polynucleotide encoding the cytoplasmic domain of FcyR. In certain embodiments, the polynucleotide encoding the cytoplasmic domain of Dectin- 1 is downstream the polynucleotide encoding the cytoplasmic domain of FcyR.

[0091] The cytoplasmic domain of Dectin-1 may comprise an amino acid sequence set forth in SEQ ID NO: 1, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof.

[0092] The cytoplasmic domain of FcyR may comprise an amino acid sequence set forth in SEQ ID NO: 2, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof.

[0093] In certain embodiments, the intracellular signaling domain of the CAR described herein comprises an amino acid sequence set forth in SEQ ID NO: 3, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof.

[0094] In certain embodiments, the intracellular signaling domain of the CAR described herein comprises an amino acid sequence encoded by a nucleic acid sequence set forth in SEQ ID NO: 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or any functional forms thereof.

[0095] (4) Other regions

[0096] In some embodiments, the CAR further comprises a signal peptide. In some embodiments, the signal peptide comprises a signal peptide of CD 8 alpha. In some embodiments, the signal peptide of CD8 alpha comprises a sequence of SEQ ID NO: 5, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto while retaining substantial biological activity thereof, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereof, or any functional forms thereof.

[0097] (5) Source of Cells

[0098] The modified immune cell provided herein may be obtained from any source. In certain embodiments, the modified immune cell provided herein is derived from immune cells isolated from subjects, e.g., human subjects. In some embodiments, the immune cells are obtained from a subject of interest, such as a subject suspected of having a cancer, a subject suspected of having a predisposition to a cancer, a subject who will undergo, is undergoing, or have undergone treatment for a cancer, a subject who is a healthy volunteer or healthy donor, or from blood bank. In some embodiments, the immune cells are obtained from a cancer subject who has poor responsiveness to an anti-cancer agent comprising a small molecule, such as chemotherapy or antibody drug conjugate (ADC). In some embodiments, the immune cells are obtained from humanized mice.

[0099] In some embodiments, the immune cell is selected from the group consisting of: a dendritic cell, a T cell, a Natural Killer (NK) cell, a NKT cell, a B cell, a macrophage cell, an eosinophil or a neutrophil. In certain embodiments, the immune cell is a dendritic cell or a precursor or progenitor cell thereof. The term “dendritic cell or a precursor or progenitor cell thereof, as used herein, refers to a native or modified dendritic cell or a precursor or progenitor cell thereof.

[0100] The cells can be autologous or allogeneic to the subject of interest. Allogeneic donor cells may not be human-leukocyte-antigen (HLA)-compatible, and thus allogeneic cells can be treated to reduce immunogenicity.

[0101] Immune cells can be collected from any location in which they reside in the subject including, but not limited to, blood, cord blood, spleen, thymus, lymph nodes, pleural effusion, spleen tissue, tumor and bone marrow. The isolated immune cells may be used directly, or they can be stored for a period of time, such as by freezing.

[0102] In some embodiments, the modified immune cells are obtained by engineering a dendritic cell or a precursor or progenitor cell thereof. A dendritic cell or a precursor or progenitor cellthereof can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as apheresis. In some embodiments, the dendritic cell or a precursor or progenitor cell thereof is derived from peripheral blood cells (e.g., peripheral blood mononuclear cells, such as a monocyte), a bone marrow cell, an embryonic stem cell, or an induced pluripotent stem cell (iPSC).

[0103] The presence of a dendritic cell can be checked using the previously described method. For example, a dendritic cell may be identified by measuring expression of CDllc, CD80, CD86, MHC / HLA molecules, and / or CCR7 molecules, which can be detected using techniques, such as immune chemistry, immunophenotyping, flow cytometry, Elispots assays, classical tetramer staining, and intracellular cytokine staining.

[0104] The modified immune cells provided herein can be produced using methods as described in PCT / CN2021 / 141311, the entire disclosure of which is incorporated herein by reference. In some embodiments, the method comprises introducing to a starting cell the vector comprising the polynucleotide encoding the CARs provided herein under conditions suitable for expression of the polynucleotide. The method provided herein may comprise one of more steps selected from: obtaining a starting cell (i.e., a cell from a source), culturing (including expanding, optionally including maturating) the starting cell, and genetically modifying the cells. The starting cell can be a dendritic cell or a precursor or a progenitor cell thereof as described above.

[0105] Genetically modifying a DC or a precursor or progenitor cell thereof can be accomplished by transducing a population of substantially homogeneous DCs with a polynucleotide encoding a CAR provided herein. In certain embodiments, a retroviral vector (e.g., a lentiviral vector) is employed for the introduction of the polynucleotide provided herein into the DCs. For example, the polynucleotide provided herein can be cloned into a lentiviral vector and expression can be driven from its endogenous promoter, from the lentiviral long terminal repeat, or from a promoter specific for a target cell type of interest. Common delivery methods for delivering viral vectors include but is not limited to, electroporation, microinjection, gene gun, and magnetofection. Placement of a presently disclosed CAR can be made at any endogenous gene locus.

[0106] In certain embodiment, the modified immune cells provided herein are prepared by transfecting polynucleotide encoding the CARs provided herein into a DC prior to administration. In certain embodiments, the engineered cell provided herein can be made by transfecting a precursor or progenitor cell of DC with the polynucleotide encoding the CARs provided hereinvia, for example, a viral vector, followed by differentiating the transfected cell into a DC. The engineered cells provided herein exhibit improved expression of CARs on the cell surface. The precursor or progenitor cell of a DC can be derived from peripheral blood cells (e.g., peripheral blood mononuclear cells, such as a monocyte, e.g., THP-1 cell, peripheral monocytes), a bone marrow cell. The precursor or progenitor cell of a DC can also be an embryonic stem cell, or an induced pluripotent stem cell (iPSC).

[0107] The modified immune cell comprises a polynucleotide encoding a CAR capable of activating a dendritic cell can be identified and selected using the method as described in PCT / CN2021 / 141311 , the entire disclosure of which is incorporated herein by reference. In certain embodiments, the modified immune cell comprises a polynucleotide encoding a CAR capable of activating a dendritic cell can be identified and selected by providing a non-human animal comprising an immune suppressive tumor microenvironment. In certain embodiments, the immune suppressive tumor microenvironment is clinically relevant. As used herein, The term “clinically relevant” with respective to the immune suppressive tumor microenvironment or tumor immune suppressive microenvironment (TIME) refers to an immune suppressive tumor microenvironment characterized in one or more of the following features: 1) hypoxic and acidic, 2) enriched with negative immune regulatory cells, such as regulatory T cells, immune suppressive DC cells, tumor associated macrophage and tumor associated fibroblasts, 3) with the overexpression of immune suppressive molecules, such as PD-1, TIM3, TIGIT, LAG3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR(CD155) and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR(CD155), HLA class I, sialoglycoprotein, CD112, CD113, Galectin9, CD24, and CD47; and 4) capable of suppressing the activities of the tumor-infiltrating immune cells (e.g., immune effector cells). “Effector cells” used in the context of immune cells refers to cells that can be activated to carry out effector functions in response to stimulation. Effector cells may include, without limitation, NK cells, cytotoxic T cells and helper T cells.

[0108] In certain embodiments, the non-human animal (e.g., mouse) model comprises human fetal thymus and autologous human hematopoietic stem cells (e.g., autologous human CD34+ hematopoietic stem cells, for example, autologous human fetal liver CD34+ hematopoietic stem cells). The term “autologous” as used herein may refer to that the human hematopoietic stem cells and the human fetal thymus are generated from the same fetal source. In certain embodiments, thenon-human animal (e.g., mouse) model is injected with about I xlO5to about 5xl05autologous human hematopoietic stem cells (e.g., autologous human CD34+ hematopoietic stem cells, for example, autologous human fetal liver CD34+ hematopoietic stem cells). In certain embodiments, the non-human animal (e.g., mouse) model comprises a sustained human immune system comprising human lymphohematopoietic cells, such as T cells (e.g., CD3+T cells), B cells (e.g., CD19+B cells) and optionally dendritic cells (DCs), which allows normal human T cell maturation in the presence of autologous human leukocyte antigens (HLAs) inside a human thymus environment. In certain embodiments, the non-human animal is a rodent, such as a rat or a mouse.

[0109] In certain embodiments, the non-human animal comprises an immune suppressive microenvironment, for example, an immune suppressive tumor microenvironment. In certain embodiments, the immune suppressive tumor microenvironment comprises a tumor and / or tumor infiltrating immune cells expressing an immune inhibitory molecule. The immune inhibitory molecule can be selected from the group consisting of PD-1, TIM3, TIGIT, LAG3, A2AR, BTLA (CD272), CTLA-4 (CD 152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR(CD155) and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR(CD155), HLA class I, sialoglycoprotein, CD112, CD113, Galectin9, CD24, and CD47. In certain embodiments, the immune inhibitory molecule is CTLA-4 and / or PD-L1. In certain embodiments, the tumor comprises a cell expressing CTLA4-Ig and / or PD-L1.

[0110] The method provided herein further comprises: administering a dendritic cell expressing a candidate CAR to the non-human animal described above, detecting a marker for the dendritic cell activation that comprises, for example, improved infiltration to the immune suppressive tumor microenvironment, improved survival rate, and / or enhanced function in inducing activation of immune cells (e.g., T cell, a Natural Killer (NK) cell, a NKT cell, a B cell, a macrophage cell, an eosinophil or a neutrophil) when compared to a reference DC, and selecting the candidate CAR as a CAR capable of activating DCs. In certain embodiments, the immune cell is a T cell selected from the group consisting of CD4+ T cell, CD8+ T cell, cytotoxic T cell, terminal effector T cell, memory T cell, naive T cell, natural killer T cell, gamma-delta T cell, cytokine-induced killer (CIK) T cell, and tumor infiltrating lymphocyte. In certain embodiments, the immune cell is autologous or allogeneic. In certain embodiments, the immune cell is a modified immune cell (e.g., CAR-T cells) or a native immune cell. In certain embodiments, the modified immune cell (e.g., CAR-T cells) is administered in combination with the dendritic cell expressing the candidate CAR.[oni] The method of selecting DC-activating CARs involving a non-human animal with clinically relevant TIME provides more clinically relevant DC-activating CARs. In other words, the DC-activating CARs selected by the method provided herein can not only activate DCs in an animal model but can also be expected to activate DCs under clinical settings, which can hardly be achieved so far by conventional animal models due to the increased complexity and heterogeneity of the tumor microenvironment in human patients as compared to the conventional animal models.

[0112] 2 Anti-cancer Agent

[0113] In some embodiments, the anti-cancer agent provided herein comprises a small molecule drug or an antibody-drug conjugate (ADC). In some embodiments, the anti-cancer agent is a small molecule drug. In some embodiments, the anti-cancer agent is an antibody drug conjugate (ADC). In some embodiments, the anti-cancer agent is a chemotherapy drug. As used herein, the term “chemotherapy drug” refers to a class of small-molecule drugs that can kill fast-growing cells, such as cancer cells. The “small molecule” as used herein refers to a compound having a molecular weight of less than or equal to about 2 kDa. In some embodiments, the small molecule has a molecular weight of less than or equal to about 1.5 kDa. In some preferred embodiments, the small molecule has a molecular weight of less than or equal to about 1 kDa, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400Da, 300 Da, 200 Da or 100 Da. As used herein, the term “antibody drug conjugate” or “ADC” refers to a class of biopharmaceutical drugs that combine the specificity of monoclonal antibodies (mAbs) with the potent cytotoxicity of small-molecule drugs. ADCs are designed to deliver cytotoxic payloads directly to tumor cells, minimizing systemic exposure and toxicity. In an ADC, generally, a mAb is linked to a small-molecule drug through a chemical linker. The mAb serves as a targeting agent, binding specifically to antigens expressed on the surface of tumor cells. Once bound to the tumor cell, the ADC is internalized into the cell, where the linker is cleaved to release the cytotoxic payload. The released drug then exerts its cytotoxic effects on the tumor cell, leading to cell death.

[0114] In some embodiments, the chemotherapy drug is selected from the group consisting of: a platinum-based chemotherapy drug, a cell cycle-specific antineoplastic agent, an antibiotic chemotherapy drug, a phytokaloids chemotherapy drug (e.g., taxane), a targeted small molecule inhibitor and an anti-metabolite chemotherapy drug. In some embodiments, the chemotherapy drugis selected from the group consisting of: etoposide, cisplatin, gemcitabine, docetaxel, paclitaxel, Cyclophosphamide, Doxorubicin, 5 -Fluorouracil, Capecitabine, Vincristine, Erlotinib, Gefitinib, Afatinib, Irinotecan, Irinotecan, Bevacizumab, Cetuximab, Sorafenib, Abiraterone, Carbop latin, Bevacizumab, Temsirolimus, Sorafenib, Palbociclib, Trastuzumab and Dacarbazine. In some embodiments, the ADC is selected from the group consisting of: Trastuzumab deruxtecan (DS- 8201a, T-DXd), Sacituzumab govitecan, Trastuzumab Emtansine (T-DM1), Patritumab deruxtecan, Vorsetuzumab mafodotin, Anetumab-MMAEClivatuzumab-MMAE, Indusatumab vedotin, Lifastuzumab vedotin, Anetumab ravtansine, Polatuzumab vedotin-piiq, Zilovertamab vedotin, Farletuzumab-MMAE, Iladatuzumab vedotin, Pinatuzumab vedotin, Sirtratumab vedotin, Codrituzumab-MMAE, Cetuximab-MMAE, Tusamitamab ravtansine, Serclutamab talirine, Vadastuximab talirine, Cantuzumab mertansine, Cantuzumab ravtansine, Ozuriftamab vedotin, Lorvotuzumab mertansine, Sofituzumab vedotin, Laprituximab emtansine, Enapotamab vedotin, Praluzatamab ravtansine, Depatuxizumab mafodotin, Brentuximab vedotin, Telisotuzumab vedotin, Labetuzumab govitecan, Disitamab vedotin, Datopotamab deruxtecan, Tisotumab vedotin, Trastuzumab MMAE, Enfortumab vedotin-ejfv, Glembatumumab vedotin, Loncastuximab tesirine, Ladiratuzumab vedotin, IMGN779, Anti-FOLRl (mirvetuximab-MMAE), Anti-GPC3 / Glypican-3 (Codrituzumab-MMAE), Anti-KAAGl (ADCT-901-MMAE), Sacituzumab- govitecan and Trastuzumab-Emtansine (T-DM1).

[0115] In some embodiments, the anti-cancer agent comprises a platinum-based chemotherapy drug, and the cancer is glioma. In some embodiments, the anti-cancer agent comprises a cell cyclespecific antineoplastic agent, and the cancer is colorectal cancer or liver cancer. In some embodiments, the anti-cancer agent comprises an antibiotic chemotherapy drug or an ADC drug, and the cancer is breast cancer or ovarian cancer. In some embodiments, the anti-cancer agent comprises a phytokaloids chemotherapy drug or an ADC drug, and the cancer is lung cancer. In some embodiments, the anti-cancer agent comprises an ADC drug, and the cancer is pancreatic cancer.

[0116] III. Kit

[0117] In one aspect, the present disclosure provides a kit comprising (a) a first composition comprising a population of modified immune cells, and (b) a second composition comprising an anti-cancer agent, wherein the modified immune cell comprises a polynucleotide encoding achimeric antigen receptor (CAR) capable of activating a dendritic cell. In certain embodiments, the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR. In certain embodiments, the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

[0118] In some embodiments, the first composition and the second composition are in separate containers.

[0119] In another aspect, the present disclosure provides a kit comprising (a) a first composition comprising a polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell, and (b) a second composition comprising an anti-cancer agent. In certain embodiments, the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR. In certain embodiments, the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

[0120] In some embodiments, the first composition and the second composition are in separate containers.

[0121] In one aspect, the present disclosure provides a kit comprising a pharmaceutical composition comprising a population of modified immune cells, and an insert comprising administering the pharmaceutical composition in combination with an anti-cancer agent, wherein the modified immune cell comprises a polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell. In certain embodiments, the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR. In certain embodiments, the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

[0122] In another aspect, the present disclosure provides a kit comprising a polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell, and an insert comprising administering an effective amount of modified immune cells comprising the polynucleotide in combination with an anti-cancer agent. In certain embodiments, the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmicdomain of Dectin- 1 and a cytoplasmic domain of FcyR. In certain embodiments, the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

[0123] The structures of modified immune cells and the CARs provided in this section have been described under the section “1. Modified Immune Cell” above.

[0124] In some embodiments, the kits of the present disclosure comprise written instructions for the use of the kit. In certain embodiments, the instructions include at least one of the following: clinical studies, precautions, warnings, and / or references. The instructions can be either printed directly on the container (when present) or provided in the container or with the container as a label applied to the container, or as a separate sheet, pamphlet, card, or folder. Suitable containers include, for example, bottles, syringes, vials, and test tubes. The containers can be formed from a variety of materials such as plastic or glass. In certain embodiments, the container holds the pharmaceutical composition provided herein and have a sterile access port.

[0125] In certain embodiments, the kit further comprises a second container comprising a pharmaceutically acceptable medium as described above. In certain embodiments, the kit further comprises other materials that are commercially desirable or user friendly, such as other diluents, buffers, needles, filters, syringes, and package inserts with instructions for use.EXAMPLES

[0126] Example 1. Preparation of humanized mouse tumor model and generation of modified dendritic cells (DCs)

[0127] This example illustrates the preparation of humanized mouse tumor model and generation of modified DCs.

[0128] 1.1 Generation of humanized mouse tumor model

[0129] A humanized model, in which human solid tumors developed clinically relevant tumor immune suppressive microenvironment (TIME) in the immune system humanized mice (Hu-mice) was generated as previously described (0. Li, et al., Developing Covalent Protein Drugs via Proximity-Enabled Reactive Therapeutics. Cell 182, 85-97.el6 (2020)). In brief, NOD / SCID / IL- 2y- / - (NCG) mice, purchased from Gempharmatech Co., Ltd, were transplanted with human fetal thymus and CD34+ cells. As such, Hu-mice were established, which were also used to inoculatehuman tumors to establish humanized mouse tumor model (Hu-mice tumor model) used in the present disclosure.

[0130] 1.2 Generation of modified DCs

[0131] This example illustrates the preparation of modified DCs.

[0132] DCs were derived from the bone marrow cells of the Hu-mice. Specifically, the femur and tibia of a humanized mouse were sterilely excised, disinfected in 70% alcohol, and rinsed with cold PBS. Bone marrow cells were flushed, dispersed, filtered, and centrifuged. After lysing red blood cells, the remaining cells were washed, counted, and cultured at 1 * 106 / ml in a differentiation medium with GM-CSF and IL-4, replenishing the medium every two days.

[0133] On the 8thday of differentiation, immature DCs were infected with the lentivirus encoding the chimeric antigen receptor as illustrated in FIG. 1 with an MOI of 2-20. The amino acid sequence of scFv is set forth in SEQ ID NO: 16. The amino acid sequence of hinge is set forth in SEQ ID NO: 7. The amino acid sequence of the transmembrane domain (TM) is set forth in SEQ ID NO: 6. The amino acid sequence of the cytoplasmic domain (Dec + Fcr) is set forth in SEQ ID NO: 3. The lentiviral stock was thawed, mixed with protamine sulfate, and added to differentiation medium. After incubating for 12 hours, more medium was added. Cells were collected 24 hours later, washed, and cultured until day 10 and modified DCs were obtained.

[0134] Example 2. Combination of modified DCs with platinum-based chemotherapy drugs for treating glioma

[0135] This example illustrates the combination of the modified DCs and platinum-based chemotherapy drugs in treating glioma in a Hu-mice xenograft model.

[0136] The method described in Example 1 was used to established humanized mice and to prepare modified dendritic cells (DCs).

[0137] After harvesting U87 cells with trypsin, the cells were then resuspended in PBS-Matrigel buffer. The back hair of humanized mice was shaved off, and the glioblastoma cells were transplanted under the skin of the mice. After the tumor was visible, the size of the tumor was measured. The mice were divided into two groups, and cisplatin (lOmg / ml, 50ul) or PBS was injected into the tumors of the mice. Two days later, the mice were further divided into 4 groups, modified DCs (2 / 106cells / mouse) or PBS buffer were injected into the mice via the tail vein. The growth of the tumors was continuously monitored, and the size of the tumors was measuredregularly. The tumor volume was calculated using the formula: tumor volume = (width2x length) x 0.5.

[0138] From the growth curve in FIG. 2, it can be seen that the modified DCs provided herein could effectively enhance the antitumor effect of cisplatin on human glioblastoma in Hu-mice, inhibiting the growth of glioblastoma. The statistical analysis (t-Test) of tumor volume in each group showed significant differences between groups, denoted by: * p<0.05, ** p<0.01, *** p<0.001, **** pO.OOOl.

[0139] Example 3. Combination of modified DCs with cell cycle-specific antineoplastic agents for treating colorectal cancer

[0140] This example illustrates combination of the modified DCs and cell cycle-specific antineoplastic agents in treating colorectal cancer in a Hu-mice xenograft model.

[0141] The method described in Example 1 was used to established humanized mice and to prepare modified dendritic cells (DCs).

[0142] After digestion with pancreatin, the colorectal cancer cell line HCT16 cells were resuspended in PBS-Matrigel solution. The back hair of humanized mice was shaved off, and then the colorectal cancer cells were transplanted subcutaneously into the mice. After the tumors in the mice became visible, the tumor size was measured. The mice were divided into two groups, and the tumors in each group were injected with either lOmg / ml of Etoposide (50ul) or PBS. After two days of treatment, each group of mice was further divided into 4 subgroups. Subsequently, the modified DCs (2xl06cells / mouse) provided herein or PBS buffer were injected into the mice via the tail vein. The tumor growth in the mice was continuously monitored, and the tumor size was measured regularly. The tumor volume was calculated using the following formula: tumor volume = (width2x length) x 0.5.

[0143] FIG. 3 shows the tumor growth curve of humanized mice with colorectal cancer cells HCT116 treated with the modified DCs provided herein in combination with the chemotherapy drug Etoposide. From the growth curve, it was evident that the modified DCs provided herein could effectively enhance the anti-tumor effect of Etoposide on humanized mice with colorectal cancer, inhibiting tumor growth. The statistical analysis of tumor volume among the groups demonstrated significant differences, with * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0144] Example 4. Combination of modified DCs with cell cycle-specific antineoplastic agents for treating liver cancer

[0145] This example illustrates combination of the modified DCs provided herein and cell cyclespecific antineoplastic agents in treating liver cancer in a Hu-mice xenograft model.

[0146] The method described in Example 1 was used to established humanized mice and to prepare modified dendritic cells (DCs).

[0147] After digesting HepG2 liver cancer cells with pancreatin, the cells were resuspended in PBS-Matrigel solution. The back hair of humanized mice was shaved off and the liver cancer cells were then transplanted under the skin of the mice. Once tumors became visible, they were measured. The mice were divided into two groups, and either lOmg / ml, 50ul of Etoposide or PBS was injected into the tumor. Two days after treatment, each group of mice was further divided into four groups, and either modified DCs (2 / 106cells / mouse) or PBS buffer was intravenously injected into the mice's tail veins. After cell injection, the growth of the tumors in the mice was continuously monitored, and the tumor sizes were measured regularly. The tumor volume was calculated using the following formula: tumor volume = (width2x length) x 0.5.

[0148] FIG. 4 shows the tumor growth curves of the modified DC cells provided herein combined with the chemotherapy drug Etoposide, in treating HepG2 human liver cancer cells in a humanized xenograft mouse model. From the growth curves, it could be observed that the modified DC cells provided herein effectively enhance the anti-tumor effect of Etoposide on the xenograft liver cancer, inhibiting tumor growth. The statistical analysis (t-Test) of the tumor volumes among the groups demonstrates significant differences between the groups: * p<0.05, ** p<0.01, *** p<0.001, **** pO.OOOl.

[0149] Example 5. Combination of modified DCs with antibiotic chemotherapy drugs for treating breast cancer

[0150] This example illustrates combination of the modified DCs provided herein and antibiotic chemotherapy drugs in treating breast cancer in a Hu-mice xenograft model.

[0151] The method described in Example 1 was used to established humanized mice and to prepare modified dendritic cells (DCs).

[0152] Breast cancer cell line MCF7 cells are digested using trypsin and then resuspended in PBS- Matrigel solution. The back hair of humanized mice was shaved off and the breast cancer cells were then transplanted under the mice's skin. Once the tumors are visible, the mice are divided into two groups. The tumors in each group are injected with either Doxorubicin (0.4mg / ml, 50ul) or PBS intratumorally. Two days after treatment, Doxorubicin group of mice was further divided into two groups, and either 2E+06 modified DC cells or PBS buffer was intravenously injected into the mice's tail veins. The growth of the mouse tumors is continuously monitored, and the tumor size is measured regularly. The tumor volume is calculated using the following formula: tumor volume = (width2x length) x 0.5.

[0153] FIG. 5 shows the tumor growth curve of humanized mice with breast cancer cell MCF7 treated with the modified DC cells provided herein in combination with the chemotherapy drug Doxorubicin. From the growth curve, it could be seen that the modified DCs provided herein could effectively enhance the anti-tumor effect of Doxorubicin on humanized mouse breast cancer, inhibiting tumor growth. The statistical analysis (t-test) demonstrates significant differences between the groups in terms of tumor volume, with * p<0.05, ** p<0.01, *** p<0.001, and **** pO.OOOl.

[0154] Example 6. Combination of modified DCs with phytokaloids chemotherapy drugs for treating lung cancer

[0155] This example illustrates combination of the modified DCs provided herein and phytokaloids chemotherapy drugs in treating lung cancer in a Hu-mice xenograft model.

[0156] The method described in Example 1 was used to established humanized mice and to prepare modified dendritic cells (DCs).

[0157] After digestion with trypsin, A549 lung cancer cells were resuspended in PBS-Matrigel solution. The back hair of humanized mice was shaved off, and the lung cancer cells were then transplanted subcutaneously into the mice. Once the tumors became visible, the mice were measured and divided into two groups. The tumors in each group were injected with either paclitaxel (2mg / ml, 50ul) or PBS. After three days of treatment, the groups of mice were further divided into four groups. Each group received intravenous injection of modified DC cells (3xl06cells per mouse) or PBS buffer. The growth of the tumors in the mice was continually monitored,and the tumor size was measured regularly. The tumor volume was calculated using the following formula: tumor volume = (width2x length) x 0.5.

[0158] FIG. 6 shows the tumor growth curve of humanized mice with lung cancer cell line A549 treated with the modified DCs provided herein and chemotherapy drug paclitaxel. From the growth curve, it could be observed that the modified DCs provided herein could effectively enhance the anti-tumor effect of paclitaxel on humanized mouse lung cancer, inhibit tumor growth. The statistical analysis of tumor volumes between groups using t-Test shows significant differences: * p<0.05, ** p<0.01, *** p<0.001, **** pO.OOOl .

[0159] Example 7. Combination of modified DCs with phytokaloids chemotherapy drugs for treating liver cancer

[0160] This example illustrates combination of the modified DCs provided herein and phytokaloids chemotherapy drugs in treating liver cancer in a Hu-mice xenograft model.

[0161] The method described in Example 1 was used to established humanized mice and to prepare modified dendritic cells (DCs).

[0162] After digestion with trypsin, Huh7 liver cancer cells were resuspended in PBS-Matrigel solution. Immune system humanized mice are prepared by shaving the hair on the back, and the liver cancer cells were then transplanted subcutaneously into the mice. Once the tumors became visible, the mice were measured and divided into two groups. The tumors in each group were injected with either paclitaxel (2mg / ml, 50ul) or PBS. After three days of treatment, the groups of mice were further divided into four groups. Each group received intravenous injection of modified DC cells (3xl06cells per mouse) or PBS buffer. The growth of the tumors in the mice was continually monitored, and the tumor size was measured regularly. The tumor volume was calculated using the following formula: tumor volume = (width2x length) X 0.5.

[0163] Figure 7 shows the tumor growth curve of humanized mice with liver cancer cell line Huh7 treated with the modified DCs provided herein and chemotherapy drug paclitaxel. From the growth curve, it could be observed that treating with modified DC cells, paclitaxel or combination could effectively provide the anti-tumor effect.

[0164] Example 8. Combination of modified DCs with ADC drugs for treating ovarian cancer

[0165] This example illustrates combination of the modified DCs and ADC drugs in treating ovarian cancer in a Hu-mice xenograft model.

[0166] The method described in Example 1 was used to established humanized mice and to prepare modified dendritic cells (DCs).

[0167] Ovarian cancer cells (e.g., SKOV3) were transplanted subcutaneously into humanized mice. Once the tumor reached a visible size, the mice were divided into groups and treated with either PBS or the ADC drug (Trastuzumab deruxtecan, Selleckchem, ~3mg / kg). After 3 days of drug administration, the modified DCs (1.25*106cells) provided herein or PBS were introduced for continued treatment. The growth of the tumors in the mice was continually monitored, and the tumor size was measured regularly. The tumor volume was calculated using the following formula: tumor volume = (width2x length) x 0.5. The growth curve was formed by dividing the tumor volume at each time point by the tumor volume at Day 0. To evaluate the levels of cytokines post treatment with CARDC and ADC therapy, we collected serum samples from mice before and after drug administration. We used the Beadstar-mPlex assay kit to monitor multiple cytokines. The experiment was conducted according to the instructions provided in the kit manual. First diluted the standard product 4 times to create 7 concentrations ranging from 5000pg / ml to 2.4pg / ml, with a blank well concentration of 0. Dilute the serum samples 2-4 times and set aside. Took a clean 96-well U-shaped (or V-shaped) cell culture plate and add the standard solution or test sample solution. Vortex mixed the A solution (fluorescent microsphere mixed with cross-linked antibody) thoroughly to disperse the microspheres evenly. Added 50pL of A solution to each well containing sample or standard, mixed well, and shook at 25°C for 1.5 hours (200 rmp / min). After the first antibody incubation, centrifuged the plate at 500g for 5 minutes and discarded all supernatant. Added 200pL washing solution to each well, centrifuged at 500g for 5 minutes, discarded all supernatant, repeated twice. Vortex mixed the B solution (mixed detection antibodies) thoroughly, then added 50pL of B solution to each well containing sample or standard, mixed well, and shook at 25°C for 1 hour (200 rmp / min). After the second antibody incubation, added 200pL washing solution to each well, centrifuged at 500g for 5 minutes, discarded all supernatant. Added 200pL washing solution to each well, centrifuged at 500g for 5 minutes, discarded all supernatant. Added lOOpL of E solution to each well, shook well to transfer the microspheres in the well to a flow tube or directly detect on the machine (flow cytometer with a 96-well plate sampler). Collected 100- 200 particles of each factor. Circled the valid particle group in the FSC-A and SSC-A plots, thendisplayed the corresponding target microsphere group in the APC-A and APC-Cy7-A channels within the circled group. Displayed a histogram of the target microsphere group, with PE-H on the x-axis representing the average fluorescence intensity (MFI). In an Excel sheet, took the logarithm (1g) of the MFI values of the collected samples and standards. Plotted the 1g MFI values against the 1g concentrations of the standard solutions and performed linear fitting to obtain a first-degree equation. Substituted the 1g MFI value of the sample into the equation to calculate the 1g value of the concentration of the target factor in the sample. Calculated the exponential value of 10, which represented the concentration of the target cell factor in the sample after dilution, then multiplied by the dilution factor of the sample to obtain the concentration of the target cytokine in the sample.

[0168] FIG. 8 showed the tumor growth curve of humanized mice with ovarian cancer cell line SKOV3 treated with the modified DCs provided herein and ADC drug Trastuzumab deruxtecan. From the growth curve, it could be observed that the modified DCs provided herein could effectively enhance the anti -turn or effect of Trastuzumab deruxtecan on humanized mouse ovarian cancer, inhibit tumor growth. The statistical analysis of tumor volumes between groups using t- Test shows significant differences: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0169] As shown in FIG.9, which illustrates the levels of cytokines after treatment with CARDC and ADC, the use of CARDC, ADC, or both together does not trigger a cytokine storm.

[0170] Example 9. Combination of modified DCs with ADC drugs for treating breast cancer

[0171] This example illustrates combination of the modified DCs and ADC drugs in treating breast cancer in a Hu-mice xenograft model.

[0172] The method described in Example 1 was used to established humanized mice and to prepare modified dendritic cells (DCs).

[0173] Breast cancer cells (e.g., SK-BR-3) are transplanted subcutaneously into humanized mice. Once the tumor reaches a visible size, the mice are divided into groups and treated with either PBS or the ADC drug (Trastuzumab deruxtecan, Selleckchem,~3mg / kg). After 2 days of drug administration, the modified DCs (~2*106cell) provided herein or PBS are introduced for continued treatment. The growth of the tumors in the mice was continually monitored, and the tumor size was measured regularly. The tumor volume was calculated using the following formula: tumor volume = (width2x length) x 0.5.

[0174] FIG. 10 showed the tumor growth curve of humanized mice with breast cancer cell line SKBR3 treated with the modified DCs provided herein and ADC drug Trastuzumab deruxtecan.From the growth curve, it could be observed that the modified DCs provided herein could effectively enhance the anti-tumor effect of Trastuzumab deruxtecan on humanized mouse breast cancer, inhibit tumor growth. The statistical analysis of tumor volumes between groups using t- Test shows significant differences: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0175] Example 10. Combination of modified DCs with ADC drugs for treating lung cancer

[0176] This example illustrates combination of the modified DCs provided herein and ADC drugs in treating lung cancer in a Hu-mice xenograft model.

[0177] The method described in Example 1 was used to established humanized mice and to prepare modified dendritic cells (DCs).

[0178] Lung cancer cells (e.g., A549) were transplanted subcutaneously into humanized mice. Once the tumor reached a visible size, the mice were divided into groups and treated with either PBS or the ADC drug (Cetuximab-MMAE, Selleckchem or Trastuzumab deruxtecan, Selleckchem). After a few days of drug administration, the modified DCs provided herein or PBS were introduced for continued treatment. Tumor growth was continuously monitored by regularly measuring the tumor size. The tumor volume was calculated using the following formula: tumor volume = (width2x length) x 0.5. The growth curve was formed by dividing the tumor volume at each time point by the tumor volume at Day 0.

[0179] FIG. 11 showed the tumor growth curve of humanized mice with lung cancer cell line A549 treated with the modified DCs provided herein and ADC drug Trastuzumab deruxtecan. It can be seen that Trastuzumab deruxtecan treatment of Her2(-) lung cancer cells A549 did not effectively enhance its anti-tumor effect. This demonstrated the specificity of ADC combined with CARDC therapy.

[0180] Example 11. Combination of modified DCs with ADC drugs for treating pancreatic cancer

[0181] This example illustrates combination of the modified DCs provided herein and ADC drugs in treating pancreatic cancer in a Hu-mice xenograft model.

[0182] The method described in Example 1 was used to established humanized mice and to prepare modified dendritic cells (DCs), except that the scFv used in Example 1 was replaced with an scFv having the amino acid sequence set forth in SEQ ID NO: 26.

[0183] Pancreatic cancer cells (e.g., Bxcp3) were transplanted subcutaneously into humanized mice. Once the tumor reached a visible size, the mice were divided into groups and treated with either PBS or the ADC drug (TROP2-ADC, Sacituzumab govitecan, Selleckchem, Selleckchem). After drug administration, the modified DCs provided herein or PBS were introduced for continued treatment. Tumor growth was continuously monitored by regularly measuring the tumor size. The tumor volume was calculated using the following formula: tumor volume = (width2x length) x 0.5. The growth curve was formed by dividing the tumor volume at each time point by the tumor volume at Day 0.

[0184] FIG 12 showed the tumor growth curves of humanized mice bearing Bxcp3 pancreatic cancer cell lines treated with the modified DCs and ADC drug Sacituzumab govitecan (trop2- ADC) provided in this study. The growth curves demonstrated that Trop2-ADC treatment, when combined with the modified DCs described in this invention, effectively enhanced the antitumor effect and inhibits tumor growth in humanized mice bearing pancreatic cancer tumors.

[0185] Table 1 Sequences mentioned in the present disclosure

Claims

1. Claims1. A method for treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of modified immune cells in combination with an anti-cancer agent, wherein the subject comprises a cancer cell expressing a cancer surface marker, wherein the modified immune cell comprises a polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell, wherein the CAR comprises (1) an extracellular antigen-binding domain specific for the cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR, and wherein the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate (ADC).

2. The method of claim 1 , wherein the cancer is resistant or refractory to the anti-cancer agent.

3. The method of claim 1 or 2, wherein the cytoplasmic domain of Dectin- 1 comprises an amino acid sequence set forth in SEQ ID NO: 1, or any functional forms thereof.

4. The method of any one of claims 1-3, wherein the cytoplasmic domain of FcyR comprises an amino acid sequence set forth in SEQ ID NO: 2 or any functional forms thereof.

5. The method of any one of the preceding claims, wherein the intracellular signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 3 or any functional forms thereof.

6. The method of any one of the preceding claims, wherein the intracellular signaling domain comprises an amino acid sequence encoded by a nucleic acid sequence set forth in SEQ ID NO: 4 or any functional forms thereof.

7. The method of any one of the preceding claims, wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv).

8. The method of any one of the preceding claims, wherein the cancer surface marker is selected from the group consisting of: EphA2, CD19, CD70, CD133, CD147, CD171, DLL3,EGFRvIII, Mesothelin, ganglioside GD2, FAP (fibroblast activating protein), FBP (folate binding protein), Lewis Y, Claudin 18.2, IL13Ra2, HER2, MDC1, PMSA (prostate membrane specific antigen), R0R1, B7-H3, CAIX, CD133, CD171, CEA, GPC3, MUC1, CD20, BCMA, Trop2 and NKG2D.

9. The method of any one of the preceding claims, wherein the CAR further comprises a signal peptide.

10. The method of claim 9, wherein the signal peptide comprises a signal peptide of CD8 alpha.

11. The method of claim 10, wherein the signal peptide of CD8 alpha comprises an amino acid sequence set forth in SEQ ID NO: 5 or any functional forms thereof.

12. The method of any one of the preceding claims, wherein the transmembrane domain comprises a transmembrane domain of CD 8 alpha.

13. The method of claim 12, wherein the transmembrane domain of CD 8 alpha comprises an amino acid sequence set forth in SEQ ID NO: 6, or any functional forms thereof.

14. The method of any one of the preceding claims, wherein the extracellular antigen-binding domain is linked to the transmembrane domain by a hinge region.

15. The method of claim 14, wherein the hinge region comprises a hinge region of CD 8 alpha.

16. The method of claim 15, wherein the hinge region of CD8 alpha comprises an amino acid sequence set forth in SEQ ID NO: 7, or any functional forms thereof.

17. The method of any one of the preceding claims, wherein the immune cell is selected from the group consisting of: a dendritic cell, a T cell, a Natural Killer (NK) cell, a NKT cell, a B cell, a macrophage cell, an eosinophil or a neutrophil.

18. The method of claim 17, wherein the immune cell is derived from a peripheral blood cell, a bone marrow cell, an embryonic stem cell, or an induced pluripotent stem cell.

19. The method of any one of the preceding claims, wherein the cancer is a solid cancer selected from the group consisting of glioma (e.g., glioblastoma), colorectal cancer (e.g., coloncancer), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma (e.g., well-differentiated hepatocellular carcinoma)), breast cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, ovarian cancer, stomach cancer, esophageal cancer and melanoma.

20. The method of any one of the preceding claims, wherein the anti-cancer agent is a chemotherapy drug.

21. The method of claim 20, wherein the chemotherapy drug is selected from the group consisting of: a platinum-based chemotherapy drug, a cell cycle-specific antineoplastic agent, an antibiotic chemotherapy drug, a phytokaloids chemotherapy drug (e.g., taxane), a targeted small molecule inhibitor and an anti-metabolite chemotherapy drug.

22. The method of claim 20, wherein the chemotherapy drug is selected from the group consisting of: etoposide, cisplatin, gemcitabine, docetaxel, paclitaxel, Cyclophosphamide, Doxorubicin, 5 -Fluorouracil, Capecitabine, Vincristine, Erlotinib, Gefitinib, Afatinib, Irinotecan, Irinotecan, Bevacizumab, Cetuximab, Sorafenib, Abiraterone, Carboplatin, Bevacizumab, Temsirolimus, Sorafenib, Palbociclib, Trastuzumab and Dacarbazine.

23. The method of claim 1, wherein the ADC is selected from the group consisting of: Trastuzumab deruxtecan (DS-8201a, T-DXd), Sacituzumab govitecan, Trastuzumab Emtansine (T-DM1), Patritumab deruxtecan, Vorsetuzumab mafodotin, Anetumab-MMAEClivatuzumab- MMAE, Indusatumab vedotin, Lifastuzumab vedotin, Anetumab ravtansine, Polatuzumab vedotin-piiq, Zilovertamab vedotin, Farletuzumab-MMAE, Iladatuzumab vedotin, Pinatuzumab vedotin, Sirtratumab vedotin, Codrituzumab-MMAE, Cetuximab-MMAE, Tusamitamab ravtansine, Serclutamab talirine, Vadastuximab talirine, Cantuzumab mertansine, Cantuzumab ravtansine, Ozuriftamab vedotin, Lorvotuzumab mertansine, Sofituzumab vedotin, Laprituximab emtansine, Enapotamab vedotin, Praluzatamab ravtansine, Depatuxizumab mafodotin, Brentuximab vedotin, Telisotuzumab vedotin, Labetuzumab govitecan, Disitamab vedotin, Datopotamab deruxtecan, Tisotumab vedotin, Trastuzumab MMAE, Enfortumab vedotin-ejfv, Glembatumumab vedotin, Loncastuximab tesirine, Ladiratuzumab vedotin, IMGN779, Anti- FOLR1 (mirvetuximab-MMAE), Anti-GPC3 / Glypican-3 (Codrituzumab-MMAE), Anti-KAAGl (ADCT-901-MMAE), Sacituzumab-govitecan and Trastuzumab-Emtansine (T-DM1).

24. The method of any one of the preceding claims, wherein the cancer is a hematologic malignancy selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T- cell / histiocyte-rich B-cell lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma (MM).

25. The method of any one of the preceding claims, wherein: a. the anti-cancer agent comprises a platinum-based chemotherapy drug, and the cancer is glioma; or b. the anti-cancer agent comprises a cell cycle-specific antineoplastic agent, and the cancer is colorectal cancer or liver cancer; or c. the anti-cancer agent comprises an antibiotic chemotherapy drug or an ADC drug, and the cancer is breast cancer or ovarian cancer; d. the anti-cancer agent comprises a phytokaloids chemotherapy drug or an ADC drug, and the cancer is lung cancer; or e. the anti-cancer agent comprises an ADC drug, and the cancer is pancreatic cancer.

26. The method of any one of the preceding claims, wherein the effective amount of modified immune cells is administered via intravenous infusion, and / or the anti-cancer agent is administered via oral, nasal, intravenous, transdermal, subcutaneous, sublingual, or intramuscular administration.

27. The method of any one of the preceding claims, wherein the effective amount of modified immune cells is administered prior to or after the administration of the anti-cancer agent.

28. A kit comprising (a) a first composition comprising a population of modified immune cells, and (b) a second composition comprising an anti-cancer agent,wherein the modified immune cell comprises a polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell, wherein the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR, and wherein the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

29. A kit comprising (a) a first composition comprising a polynucleotide encoding a chimeric antigen receptor (CAR), and (b) a second composition comprising an anti-cancer agent, wherein the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR, and wherein the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

30. The kit of claim 28 or 29, wherein the first composition and the second composition are in separate containers.

31. A kit comprising a pharmaceutical composition comprising a population of modified immune cells, and an insert comprising instructions of administering the pharmaceutical composition in combination with an anti-cancer agent, wherein the modified immune cell comprises a polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell, wherein the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR, andwherein the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

32. A kit comprising a polynucleotide encoding a chimeric antigen receptor (CAR), and an insert comprising instructions of administering an effective amount of modified immune cells comprising the polynucleotide in combination with an anti-cancer agent, wherein the CAR comprises (1) an extracellular antigen-binding domain specific for a cancer surface marker, (2) a transmembrane domain and (3) an intracellular signaling domain comprising a cytoplasmic domain of Dectin- 1 and a cytoplasmic domain of FcyR, and wherein the anti-cancer agent comprises a small molecule drug or an antibody-drug conjugate.

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