Complement signaling as a t-cell checkpoint in the tumor microenvironment (TME)

Abstract

Provided are compositions and methods for treatment of cancer. The compositions and methods include use of a soluble recombinant complement receptor- 1 (rCR-1). The rCR-1 alone can be used as a therapeutic agent. The rCR-1 can also be used as a component of a chimeric antigen receptor (CAR) or a bi-specific binding partner such as a Bi-specific T cell engager (BiTE). Modified cells that are configured to secrete the rCR-1 are included.

Description

[0001] COMPLEMENT SIGNALING AS A T-CELL CHECKPOINT IN THE TUMOR MICROENVIRONMENT (TME)

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. provisional application no. 63 / 575,318, filed April 5, 2024, the entire disclosure of which is incorporated herein by reference.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under grants R01CA267690 and R01 CAI 88900 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 7, 2025, is named “00355101170-Sequence-Listing.xml”, and is 11,014 bytes in size.

[0008] RELATED INFORMATION

[0009] T cell checkpoint pathways are barriers to anti-tumor immunity and critical therapeutic targets. There is an ongoing and unmet need for improved compositions and methods to overcome these barriers. The present disclosure is related to this need.

[0010] BRIEF SUMMARY

[0011] The present disclosure demonstrates that complement activation in the TME delivers an inhibitory stimulus to T cells that suppresses stimulated cytokine responses required for activation and expansion.

[0012] The disclosure demonstrates that peptide C3 inhibitors (e.g., compstatins) restored CD3 / CD28-stimulated T cell cytokine responses and glucose consumption. Inhibition of Factor B, a component of the alternative pathway (AP), also rescued cytokine responses, while inhibition of C3aR, C5aR, and C7 had no significant effect. C3b / iC3b deposition on T cells was increased with ASC exposure and reduced by C3 inhibition. In illustration of an aspect of the disclosure, addition of recombinant complement receptor-1 (CR-1) fully abrogated the suppressive effect of ascites, while recombinant CD46 had variable effects among different ASC samples. Without intending to be constrained by any particular theory, it is considered that recombinant CR-1 functions as both as a decoy for membrane-bound CR- 1 signaling on T cells and inhibits complement activation by C3b and C4b binding, acting as a co-factor for Factor I-dependent cleavage of C3b and / or C4b, and C3 and C5 convertase decay acceleration function. The disclosure accordingly reveals a novel AP and CR1- dependent checkpoint of T cell activation in the TME, and provides approaches to inhibiting or preventing the T cell inhibition promoted by complement activation in the TME. Thus, in examples, a described approach comprises administering a complement receptor-1 (CR-1) to an individual.

[0013] The CR-1 may be administered as a component of a pharmaceutical formulation, or may be administered as a Bi-specific T cell engager (BiTE) format, or by way of modified cells that express and secrete the CR-1 itself or as a component of BiTE, or by using a polynucleotide that encodes the CR-1. The disclosure includes cells that are modified to produce a secretable CR-1.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the description taken in conjunction with the accompanying figures.

[0016] FIG. 1 A-1C. Data demonstrating that inhibition of complement activation by Cp40 restores cytokine secretion in T cells exposed to ascites.

[0017] FIG. 2. Data demonstrating that C3b is involved in complement-mediated inhibition of T cell cytokine responses.

[0018] FIG. 3. Data demonstrating that CR-1 (CD35) and CD46 are expressed by T cells and expression profile is not altered by stimulation, exposure to ASC or with treatment using a complement inhibitor.

[0019] FIG. 4A-4B. Data demonstrating cell surface deposition of C3b / iC3b is increased on exposure to ASC and is significantly diminished by complement inhibition.

[0020] FIG. 5A-5E. Data showing that complement-driven suppression of T cells is driven by the alternative pathway (AP) and CR-1. A-C) CD3 / CD28-stimulated normal donor T cells were cultured in media or ASC with APL-x (control), APL-38 (compstatin C3 inhibitor similar to APL-2) or APL-105 (Factor B inhibitor) for 96h, and IL-2 was measured by ELISA. APL products were provided by Apellis. C3 and factor B inhibition similarly rescued stimulated IL-2 production. Factor D inhibition with APL-107 had a similar effect (not shown) confirming the role of the alternative pathway. B) Recombinant CR-1 (rCR-1) rescued T cell IL-2 production while recombinant CD46, which also binds to and directs the cleavage of C3b toiC3b, did not. C and D) ASC increased C3b / iC3b binding to CD8+ T cells and CD4+ T cells (not shown), which was abrogated by C3 inhibition (C) and rCR-1 (D) Results are representative of at least 2 independent experiments E) rCR-1 can rescue T cell responses by functioning as a decoy for membrane-bound CR-1 signaling or through direct binding to C3b and directing its cleavage to iC3b.

[0021] DETAILED DESCRIPTION

[0022] Unless defined otherwise herein, 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 disclosure pertains.

[0023] Unless specified to the contrary, it is intended that every maximum numerical limitation given throughout this description includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. All proteins described herein include proteins that have from 90.0-99.9% identity across their entire lengths to such proteins. The amino acid or polynucleotide sequence as the case may be associated with each GenBank or other database accession number of this disclosure is incorporated herein by reference as presented in the database on the effective filing date of this application or patent.

[0024] The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.

[0025] The present disclosure relates to a model in which neutrophils are recruited to the TME where they acquire a complement-dependent T cell suppressor phenotype characterized by suppression of significant signaling and metabolic pathways necessary for T cell activation and proliferation. This model was dependent on neutrophil: T cell proximity and complement signaling inducing neutrophil suppressor function. However, substantial immune cell heterogeneity in the TME may be present, which can include niches that are T cell-rich and neutrophil-poor. The present disclosure therefore includes analysis of the role of complement-induced signaling in T cells in the TME using primary human immune cells and OC ascites fluid as authentic component of the TME. The disclosure reveals that complement signaling in T cells resulted in marked suppression of cytokine responses that was rescued by inhibition of the complement alternative pathway and the use of recombinant complement receptor-1 (CR-1). Recombinant CR-1 may function both as a decoy for membrane-bound CR-1 signaling on T cells and inhibit complement activation by C3b and C4b binding, acting as a co-factor for Factor I-dependent cleavage of C3b or C4b, and C3 and C5 convertase decay acceleration function.

[0026] The present disclosure accordingly provides compositions and methods for use in treating cancer. In examples, an agent that can specifically interact with complement or a derivative of complement is administered to an individual. The agent is configured to inhibit complement or a complement derivative from binding to membrane bound complement receptor-1 (CR-1). In an example, the agent is a complement receptor-1 (CR-1), which may be introduced into an individual using a variety of approaches as further described herein such that the CR-1 is either administered in soluble form or is expressed and optionally secreted by a modified cell that may be introduced into the individual. Without intending to be bound by any particular theory, it is considered that the CR-1 functions as a decoy of C3b binding to membrane-bound CR-1, and thereby inhibits the suppressive effect of ASC, and can also suppress complement activation by C3b and C4b binding, Factor I cofactor activity, and C3 and C5 convertase decay acceleration function.

[0027] The amino acid sequence of human CR-1 is known in the art. In an example, a CR-1 used in this disclosure and optionally modified as described herein uses the CR-1 sequence described in UniProt entry P17927, from which the amino acid sequence is incorporated herein by reference as the sequence exists in the database as of the filing date of this application. In an example, the dominant allelic variant of human CR-1 is used to provide a CR-1 in a described composition and / or method. In examples, the starting CR-1 is a 2039 amino acid protein with a 41 amino acid signal peptide, a 1930 amino acid extracellular domain, and a short cytoplasmic tail. In examples, a rCR-1 used of this disclosure lacks transmembrane and / or cytoplasmic regions. In examples, a described CR-1 comprises or consists of extracellular domains which contain short consensus repeat (SCR) domains present as long homologous repeat (LHR) domains. In examples, a rCR-1 of this disclosure comprises the amino acid sequence of a known soluble CR-1 sequence. In examples, a rCR-1 of this disclosure may be any of soluble CR-1 (sCRl) formulations, (also referred to in the art as BRL55730, TP10, and CDX-1135; or sCRl-sLex, also referred to as TP20), or APT070 (Mirococept); or CSL040, all of which are described in Hardy et al., “Soluble Complement Receptor 1 Therapeutics” Journal of Immunological Sciences, published October 21, 2022, https: / / doi.Org / 10.29245 / 2578-3009 / 2022 / 4.1240, the disclosure of which is incorporated herein by reference. In examples, the rCR-1 of this disclosure comprises an amino acid sequence of a soluble CR-1 as described in PCT publication W0201921800, the disclosure of which is incorporated herein by reference.

[0028] A representative full-length human CR-1 comprises the sequence:

[0029] MGASSPRSPEPVGPPAPGLPFCCGGSLLAVVVLLALPVAWG^CA / IPEIELPF

[0030] ARPTNLTDEFEFPIGTYLNYECRPGYSGRPFSIICLKNSVWTGAKDRCRRKSCRNPPDPVN GMVHVIKGIQFGSQIKYSCTKGYRLIGSSSATCIISGDTVIWDNETPICDRIPCGLPPTITNG

[0031] DFISTNRENFHYGSWTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIPNK CTPPNVENGILVSDNRSLFSLNEWEFRCQPGFVMKGPRRVKCQALNKWEPELPSCSRVC QPPPDVLHAERTQRDKDNFSPGQEVFYSCEPGYDLRGAASMRCTPQGDWSPAAPTCEVK SCDDFMGQLLNGRVLFPVNLQLGAKVDFVCDEGFQLKGSSASYCVLAGMESLWNSSVPV CEQIFCPSPPVIPNGRHTGKPLEVFPFGKTVNYTCDPHPDRGTSFDLIGESTIRCTSDPQG NGVWSSPAPRCGILGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCL DNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILS GNAAHWSTKPPICQRJPCGLPPTIANGDFISTNRENFHYGSWTYRCNPGSGGRKVFELVG EPSIYCTSNDDQVGIWSGPAPQCIIPNKCTPPNVENGILVSDNRSLFSLNEWEFRCQPGFV MKGPRRVKCQALNKWEPELPSCSRVCQPPPDVLHAERTQRDKDNFSPGQEVFYSCEPGY DLRGAASMRCTPQGDWSPAAPTCEVKSCDDFMGQLLNGRVLFPVNLQLGAKVDFVCDE GFQLKGSSASYCVLAGMESLWNSSVPVCEQIFCPSPPVIPNGRHTGKPLEVFPFGKAVNYT CDPHPDRGTSFDLIGESTIRCTSDPQGNGVWSSPAPRCGILGHCQAPDHFLFAKLKTQTN

[0032] ASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDI QVGSRINYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNREN FHYGSWTYRCNLGSRGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIPNKCTPPNVEN GILVSDNRSLFSLNEWEFRCQPGFVMKGPRRVKCQALNKWEPELPSCSRVCQPPPEILH GEHTPSHQDNFSPGQEVFYSCEPGYDLRGAASLHCTPQGDWSPEAPRCAVKSCDDFLGQ LPHGRVLFPLNLQLGAKVSFVCDEGFRLKGSSVSHCVLVGMRSLWNNSVPVCEHIFCPNP PAILNGRHTGTPSGDIPYGKEISYTCDPHPDRGMTFNLIGESTIRCTSDPHGNGVWSSPAPR CELSVRAGHCKTPEQFPFASPTIPINDFEFPVGTSLNYECRPGYFGKMFSISCLENLVWSSV EDNCRRKSCGPPPEPFNGMVHINTDTQFGSTVNYSCNEGFRLIGSPSTTCLVSGNNVTWD KKAPICEIISCEPPPTISNGDFYSNNRTSFHNGIWTYQCHTGPDGEQLFELVGERSIYCTS KDDQVGVWSSPPPRCISTNKCTAPEVENAIRVPGNRSFFSLTEIIRFRCQPGFVMVGSHTV

[0033] QCQTNGRWGPKLPHCSRVCQPPPEILHGEHTLSHQDNFSPGQEVFYSCEPSYDLRGAASL HCTPQGDWSPEAPRCTVKSCDDFLGQLPHGRVLLPLNLQLGAKVSFVCDEGFRLKGRSA SHCVLAGMKALWNSSVPVCEQIFCPNPPAILNGRHTGTPFGDIPYGKEISYACDTHPDRG MTFNLIGESSIRCTSDPQGNGVWSSPAPRCELSVPAACPHPPKIQNGHYIGGHVSLYLPGM TISYICDPGYLLVGKGFIFCTDQGIWSQLDHYCKEVNCSFPLFMNGISKELEMKKVYHYGD YVTLKCEDGYTLEGSPWSQCQADDRWDPPLAKCTSRTHDALIVGYLSGYIFFILLIIF

[0034] LSWIILKHRKGNNAHENPKEVAIHLHSQGGSSVHPRTLQTNEENSRVLP

[0035] (SEQ ID NO: 1).

[0036] In SEQ ID NO:1, the signal peptide is shown in bold. The extracellular domains are shown in italics and comprise the known four long homologous repeat domains (LHR-A, -B, -C, -D) as described above. The enlarged amino acids represent the transmembrane and cytoplasmic domain. In examples an rCR-1 of this disclosure may exclude some or all of the transmembrane and / or the cytoplasmic domain. In examples, a rCR-1 of this disclosure thus may comprise or consist of an amino acid segment that is precisely or approximately the 1,930 italicized amino acids of SEQ ID NO:1 or a sequence that is at least 90% identical to the italicized amino acids in SEQ ID NO:1. In examples, an rCR-1 of this disclosure sequence may be at least 90% identical to the sequence:

[0037] QCNAPEWLPFARPTNLTDEFEFPIGTYLNYECRPGYSGRPFSIICLKNSVWTGAKD RCRRKSCRNPPDPVNGMVHVIKGIQFGSQIKYSCTKGYRLIGSSSATCIISGDTVIWDNETPI CDRIPCGLPPTITNGDFISTNRENFHYGSWTYRCNPGSGGRKVFELVGEPSIYCTSNDDQ VGIWSGPAPQCIIPNKCTPPNVENGILVSDNRSLFSLNEWEFRCQPGFVMKGPRRVKCQA LNKWEPELPSCSRVCQPPPDVLHAERTQRDKDNFSPGQEVFYSCEPGYDLRGAASMRCT PQGDWSPAAPTCEVKSCDDFMGQLLNGRVLFPVNLQLGAKVDFVCDEGFQLKGSSASYC VLAGMESLWNSSVPVCEQIFCPSPPVIPNGRHTGKPLEVFPFGKTVNYTCDPHPDRGTSF DLIGESTIRCTSDPQGNGVWSSPAPRCGILGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYE CRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTT GHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSWTYRC NPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIPNKCTPPNVENGILVSDNRSLFS LNEWEFRCQPGFVMKGPRRVKCQALNKWEPELPSCSRVCQPPPDVLHAERTQRDKDN FSPGQEVFYSCEPGYDLRGAASMRCTPQGDWSPAAPTCEVKSCDDFMGQLLNGRVLFPV NLQLGAKVDFVCDEGFQLKGSSASYCVLAGMESLWNSSVPVCEQIFCPSPPVIPNGRHTG KPLEVFPFGKAVNYTCDPHPDRGTSFDLIGESTIRCTSDPQGNGVWSSPAPRCGILGHCQA PDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTP PDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRJPCGLP PTIANGDFISTNRENFHYGSWTYRCNLGSRGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQ CIIPNKCTPPNVENGILVSDNRSLFSLNEWEFRCQPGFVMKGPRRVKCQALNKWEPELPS CSRVCQPPPEILHGEHTPSHQDNFSPGQEVFYSCEPGYDLRGAASLHCTPQGDWSPEAPR CAVKSCDDFLGQLPHGRVLFPLNLQLGAKVSFVCDEGFRLKGSSVSHCVLVGMRSLWNN SVPVCEHIFCPNPPAILNGRHTGTPSGDIPYGKEISYTCDPHPDRGMTFNLIGESTIRCTSD PHGNGVWSSPAPRCELSVRAGHCKTPEQFPFASPTIPINDFEFPVGTSLNYECRPGYFGKM FSISCLENLVWSSVEDNCRRKSCGPPPEPFNGMVHINTDTQFGSTVNYSCNEGFRLIGSPS TTCLVSGNNVTWDKKAPICEIISCEPPPTISNGDFYSNNRTSFHNGTWTYQCHTGPDGEQ LFELVGERSIYCTSKDDQVGVWSSPPPRCISTNKCTAPEVENAIRVPGNRSFFSLTEIIRFRC QPGFVMVGSHTVQCQTNGRWGPKLPHCSRVCQPPPEILHGEHTLSHQDNFSPGQEVFYS CEPSYDLRGAASLHCTPQGDWSPEAPRCTVKSCDDFLGQLPHGRVLLPLNLQLGAKVSFV CDEGFRLKGRSASHCVLAGMKALWNSSVPVCEQIFCPNPPAILNGRHTGTPFGDIPYGKEI SYACDTHPDRGMTFNLIGESSIRCTSDPQGNGVWSSPAPRCELSVPAACPHPPKIQNGHYI GGHVSLYLPGMTISYICDPGYLLVGKGFIFCTDQGIWSQLDHYCKEVNCSFPLFMNGISKE LEMKKVYHYGDYVTLKCEDGYTLEGSPWSQCQADDRWDPPLAKCTSRTHD (SEQ ID N0:2).

[0038] In examples, a functional fragment of a described CR-1 is used. A “functional fragment” may be any fragment of CR-1 that is sufficient to rescue T cell responses by functioning as a decoy for membrane-bound CR-1 signaling, or that directly binds to C3b, and or facilitates cleavage of C3b to iC3b, or binds to C4b and or facilitates C4b cleavage, or exhibit decay acceleration function, or exhibit a combination of the described properties.

[0039] In examples, cells are modified to express a described CR-1 protein, such cells being suitable for use prophylactically or therapeutically. In an example, the CR-1 protein is engineered to be expressed and secreted by chimeric antigen receptor (CAR) immune cells. This approach may be used with any leukocytes that expresses the CAR (e.g., T cells, dendritic cells, Natural Killer cells, and macrophages). In an example, secretion of CR-1 may be facilitated using a sequence encoding a described CR-1 protein that is operably linked to a nuclear factor of activated T cells promoter (NF AT), which has previously been shown to promote secretion of IL-12. The sequence of the NF AT promoter is known and can be adapted to promote secretion of rCR-1 when given the benefit of this disclosure. Thus, in examples, the disclosure provides modified cells that are configured such that they secrete a described CR-1 protein. The cells may be modified such that they are engineered to bind to cancer cells and / or infiltrate a TME. In examples, a described rCR-1 protein is provided a such that it is produced and secreted from a cell that expresses a Bispecific T cell engager (BiTE). In an example, the BiTE may comprise an ScFv that inhibits binding of CR1 to its ligands (e.g., C3b or C4b). In an example, the modified cells are T cells, which may be provided as BiTE-secreting T cells (BiTE-T cells). In this regard, the disclosure includes modifying cells as described in PCT / US23 / 63359, published as WO 2023 / 164698, the disclosure of which is incorporated herein by reference. The present disclosure differs from the disclosure of WO 2023 / 164698 as far as thee BiTE-expressing cells are configured to express a described rCR-1 protein. The component of the bi-specific binding partner can target any cancer antigen, and / or can target T cells. In an example, the disclosure provides a BiTE-T cell construct that secretes a BiTE that specifically binds to a T cell antigen, such as CD3, to activate the T cell and binds to a specific tumor antigen to bring tumor cells in proximity to activated T cells; as discussed above, the BiTE-T cell can be engineered to also secrete CR-1 with the expected effect of abrogating complement-dependent signaling that impair T cell activation. In examples, modified cells of the disclosure are configured to secrete a recombinant CR-1. As such, the rCR-1 may be modified to include a secretion signal. Any suitable secretion signal can be used and many are known in the art. In non-limiting embodiments, the secretion signal comprises MALPVTALLLPLALLLHA (SEQ ID NO:3), METDTLLLWVLLLWVPGSTG (SEQ ID NO:4), MGWSCIILFLVATATGVHSD (SEQ ID NO:5), GEAAAI<EAAAI<EAAAI< (SEQ ID NO: 6) or MNSGLQLVFFVLTLKGIQG (SEQ ID NO: 7).

[0040] In examples, the other component of the bi-specific binding partner may target an antigen that is selectively expressed by ovarian cancer cells, or is overexpressed by ovarian cancer cells, relative to expression of the same target by non-cancer cells. In a non-limiting example, the bispecific binding partner binds to human Folate Receptor alpha (FRa). In examples, the bi-specific binding partner binds to a cancer antigen and binds to a CR-1 ligand, including but not necessarily limited to Peptide C3, C3b, iC3b, or a combination thereof. In examples, the BiTE is modified to include a secretion signal to combine any other BiTE component as described in WO 2023 / 164698.

[0041] The described modified T cells that express the described binding partners can be combined with any other therapeutic agent, non-limiting examples of which include conventional chemotherapeutic agents, and immune checkpoint inhibitors, the latter of which are known in the art, and target CTLA-4, PD-1, or PD-L1. Thus, the disclosure includes combination therapy using one or more described binding partners and any of CTLA-4 inhibitors, PD-1 inhibitors and PD-L1 inhibitors. As non-limiting examples, anti-PD-1 agents include Pembrolizumab and Nivolumab. Anti-PD-Ll examples include Avelumab and Atezolizumab. An anti-CTLA-4 example is Ipilimumab. The binding partners may also be combined with any other form of adoptive immunotherapy. The modified T cells may be used in autologous or allogenic therapies.

[0042] The amount of a described agent (e.g., rCR-1 itself, and any BiTE or CAR-expressing cells) used in the method can be determined by those skilled in the art, given the benefit of the present disclosure. Thus, in one embodiment, an effective amount of a composition of the invention is administered to an individual in need thereof. An effective amount can be an amount of the composition that inhibits growth of cancer cells in the individual, alleviates disease symptoms associated with the cancer, suppresses a malignant phenotype of cancer cells, inhibits growth of cancer cells, inhibits metastasis of a primary tumor, and combinations thereof. In embodiments, the individual to whom a composition of the invention is administered has, is suspected of having, or is at risk for development and / or recurrence of a cancer. In an example, the cancer is ovarian cancer, but the principles are generalizable to other tumors. In examples, the individual who is treated according to a described method has a solid tumor or a blood cancer. In examples, the individual has as stated above ovarian cancer, but may also have any of endometrial cancer, melanoma, head and neck cancer, renal cell carcinoma, breast cancer, pancreatic cancer, lung cancer, liver cancer, cervical cancer, colon cancer, esophageal cancer, glioma, glioblastoma, prostate cancer, stomach cancer, testicular cancer, bladder cancer, thyroid cancer, adrenal cancer, skin cancer, a sarcoma, leukemia, lymphoma, or myeloma.

[0043] In examples, administration of a described agent enhances the activity of an immune checkpoint inhibitor. In examples, administration of a described agent and an immune checkpoint exerts a synergistic anti-cancer response. In an example, an individual treated as described herein may have a so-called “cold” tumor. In this context, CR-1 formulations are administered to overcome complement-driven inhibition of T cell responses that are barriers to currently used immune checkpoint inhibitors and other T cell-directed cancer immunotherapy. In examples, a described composition is administered to an individual who has cancer, but does not have and / or is not at risk of developing any of: an autoimmune disease, a viral, bacterial or eukaryotic parasite infection, an inflammatory disorder, a reperfusion injury, any type of infarction, a non-cancer related ischemia, a transplantation, a neurological disorder, any type of shock. In non-limiting examples, a described rCR-1 protein may be used in conjunction with and / or to enhance the anti-cancer effects of any of PD-1 inhibitor, such as Nivolumab, Pembrolizumab, and Cemiplimab.

[0044] In non-limiting examples, a described rCR-1 protein may be used in conjunction with and / or to enhance the anti-cancer effects of any PD-L1 inhibitor, such as Atezolizumab, Avelumab, or Durvalumab.

[0045] In non-limiting examples, a described rCR-1 protein may be used in conjunction with and / or to enhance the anti-cancer effects of any CTLA-4 inhibitor, such as Ipilimumab or Tremelimumab.

[0046] In non-limiting examples, a described rCR-1 protein may be used in conjunction with and / or to enhance the anti-cancer effects of a Lymphocyte Activation Gene 3 (LAG-3) inhibitor, such as Relatlimab.

[0047] In non-limiting examples, a described rCR-1 protein may be used in conjunction with and / or to enhance the anti-cancer effects of T-cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.

[0048] The present disclosure provides a pharmaceutical composition. In examples, the pharmaceutical composition may comprise a described rCR-1 polypeptide or polynucleotide encoding the polypeptide, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition can be in the form of a liquid, e.g., a solution, emulsion, or suspension. Liquid compositions can also include one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, mono or diglycerides, polyethylene glycols, glycerin, antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pharmaceutical composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material. In an example, the described rCR-1 polypeptide is the only biological agent that is present in the pharmaceutical formulation. Pharmaceutical formulations may exclude plasma, sera, urine, blood, and other biological fluids where CR-1 may be naturally found. The described cell pharmaceutical composition may be devoid of any cell culture media or other components used to express rCR-1 for the purpose of producing the rCR-1.

[0049] Suitable routes of administrating the rCR-1 or pharmaceutical compositions described herein can include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intralesional, intraperitoneal, intraorgan, epidural, intrathecal, intramuscular, intratumoral and intravenous administrations. The disclosure includes expression vectors that encode the rCR-1, and may encode other components of CARs, BiTEs, and the like. In examples, a described construct may be delivered to cells or an individual as mRNA or DNA polynucleotides that encode the rCR-1. It is considered that administering a DNA or RNA as described herein is also a method of delivering recombinant CR-1 to an individual or to one or more cells which may be performed in vivo or ex vivo, provided the DNA is transcribed and the mRNA is translated, and / or the RNA itself is delivered and translated. Accordingly, in examples, a described rCR- 1 protein can be administered using a polynucleotide, including but not necessarily a polynucleotide comprised by any viral vector, or a polynucleotide in the form of mRNA encoding a described rCR-1 protein. In some examples, the viral vector is an adenoviral vector, lentiviral vector, retroviral vector, or adeno-associated viral vector, or a modified oncolytic virus that expresses a described rCR-1 protein.

[0050] In an example, the disclosure provides any type of eukaryotic cell that is modified to secrete rCR-1, wherein such cell is used for treating cancer in an individual,

[0051] All methods of making modified cells that are described herein and by way of the figures are included in the disclosure. In embodiments, the disclosure provides for obtaining T cells from an individual and modifying the T cells to express the described rCR-1, CARs, or BiTEs.

[0052] Examples of the disclosure is illustrated by the following statements:

[0053] Statement 1. The disclosure provides a method for treatment of cancer, the method comprising administering to an individual in need of treatment a recombinant complement receptor- 1 (rCR-1).

[0054] Statement 2. The method of Statement 1, wherein the rCR-1 inhibits binding of C3b to membrane-bound CR-1.

[0055] Statement 3. The method of Statemen 1, wherein the rCR-1 is administered as a component of a pharmaceutical formulation.

[0056] Statement 4. The method of Statement lor Statement 2, wherein rCR-1 expressed by a cell that is configured to also express a chimeric antigen receptor (CAR) or a bi-specific binding partner.

[0057] Statement 5. The method of any one of Statements 1-4, wherein the rCR-1, and the CAR or the bi-specific binding partner are expressed by a leukocyte that is optionally a T cell. Statement 6. The method of Statement 5, wherein the bi-specific binding partner is in the form of a Bi-specific T cell engager (BiTE) and wherein optionally engagement of the BiTE with a cancer cell targeted by the BiTE promotes secretion of the rCR-1.

[0058] Statement 7. The method of Statement 5, wherein the cell is configured to express the CAR, and wherein optionally engagement of the CAR promotes secretion of the rCR-1.

[0059] Statement 8. The method of any one of Statements 1-7, wherein the cancer is ovarian cancer.

[0060] Statement 9. The disclosure provides a modified leukocyte that is configured to express a chimeric antigen receptor (CAR) and rCR-1.

[0061] Statement 10. The disclosure provides a modified leukocyte that is configured to express a Bi-specific T cell engager (BiTE) and rCR-1.

[0062] Statement 11. The disclosure provides a method comprising selecting an individual who has been diagnosed with cancer, the method comprising administering to the individual a pharmaceutical composition comprising recombinant complement receptor-1 (rCR-1) or a polynucleotide encoding the rCR-1, or cells that produce the rCR-1.

[0063] Statement 12. The method of Statement 11, wherein the individual has been diagnosed with ovarian cancer.

[0064] Examples

[0065] The following Examples are intended to illustrate but not limit the disclosure. The Examples can be viewed in light of the knowledge of the skilled artisan that epithelial ovarian cancer (OC) is usually metastatic at diagnosis. Single-agent immune checkpoint inhibitors are largely ineffective in relapsed / refractory OC. Ascites is common and both the presence and volume of ascites at diagnosis are negative predictors of survival. In the examples below, we used ascites supernatants (ASC) from patients with OC as an authentic component of the TME. Complement activation products are markedly elevated in ASC versus paired serum samples. OC ASC dramatically inhibited CD3 / CD28-stimulated T cell cytokine production (IL-2, IFN-g, and IL- 10) and glucose consumption (but not proliferation) in the absence of neutrophils. The examples demonstrate that complement activation products in the TME deliver a key inhibitory stimulus to normal human T cells that suppress stimulated cytokine responses that drive T cell activation and expansion. The examples show that ASC-induced suppression of CD3 / CD28-stimulated T cell cytokine responses was fully dependent on complement activation. Addition of compstatin C3 activation inhibitors restored T cell cytokine responses and glucose consumption. Factor B inhibition also rescued cytokine responses, demonstrating that suppression was alternative pathway (AP)-dependent. Small molecule inhibitors of C3aR, C5aR, and cathepsin L (mediating intracellular C3 activation) and anti-C7 (inhibiting membrane attack complex generation) had no significant effect on T cell cytokine responses. C3b / iC3b deposition on T cells was increased with ASC exposure and reduced by C3 inhibition. As discussed above and illustrated in the figures, addition of rCR-1 completely abrogated the suppressive effect of ASC, while recombinant CD46 had variable effects among different ASC samples. Together, these results reveal a novel checkpoint of T cell activation in the TME driven by AP activation and CR-1 signaling. The disclosure therefore supports the use of CR-1 decoys, including the use of engineered T cells (e.g., CAR T-cells, BiTE T-cells) which secrete soluble CR-1 that can be expected to disrupt membrane-bound CR-1 signaling in T cells in both an autocrine and paracrine fashion. This disclosure also supports the use of antibodies, including BiTEs, which inhibit CR-1 binding to its ligands (e.g., C3b and C4b). Reflecting the known biological functions of naturally produced soluble CR-1, therapeutically administered soluble CR-1, whether administered as a recombinant protein or secreted by engineered cells, is expected to inhibit different components of complement activation that limit T cell activation in the TME. Furthermore, given the lack of data that the cytoplasmic tail of CR-1 has direct signaling function, and without intending to be bound by any particular interpretation, it is considered that upon ligation of C3b (or C4b), CR-1 binds to and induces conformational changes in proteins that leads to recruitment and activation of phosphatases that result in dephosphorylation of signaling proteins required for T cell activation. A number of CR1 -based therapeutics are in clinical development, but it is believed the present disclosure is the first to describe and provide data supporting the use of fCR-1 for treating cancer. The disclosure includes, in addition to delivery of full-length fCR-1 or functional fragments thereof as discussed that contain the C3b- or C4b-binding regions of fCR-1, the disclosure includes using adoptive cellular therapy (ACT) approaches in conjunction with CR-1 modulation. Accordingly, the disclosure includes generating tumor-infiltrating lymphocytes (TILs) with genetically engineered CR-1 that in non-limiting examples may comprise a signal peptide sequence at the N-terminus to direct it to the secretory pathway as an alternative to using rCR-1 as an isolated recombinant protein. Data described in the Examples was obtained using a commercially available rCR-1 sold as “Recombinant Human CD35 Protein” as listed under CF Catalog #: 5748-CD from R&D Systems™. The Examples are performed using human samples because of key differences between humans and mice regarding expression and function of complement proteins, including lack of CR-1 in rodents. Example 1

[0066] Example 1 is illustrated by FIGs. 1 A-1C, which provides data demonstrating that inhibition of complement activation by Cp40 restores cytokine secretion in T cells exposed to ascites. To obtain the data shown in this Example, T cells were isolated from normal donor peripheral blood, stimulated with CD3 / CD28 Dynabeads and exposed to cryopreserved ascites supernatants (ASC) obtained from newly diagnosed ovarian cancer patients. Ascites were pre-treated with compstatin (Cp40) to inhibit complement activation or scrambled peptide (SCR) as controls. Supernatants from T cell - ASC co-cultures were obtained 96hrs post activation and employed in (A) IL-2 (B) IFN-y or (C) IL- 10 ELIS As. Symbols represent individual ascites samples. Statistical comparisons made using paired t-tests.

[0067] Example 2

[0068] Example 2 is illustrated by FIG. 2 which provides data demonstrating that C3b is involved in complement mediated inhibition of T cell cytokine responses. To obtain the data shown in this Example, T cells were isolated from normal donor peripheral blood, stimulated with CD3 / CD28 Dynabeads and exposed to cryopreserved ascites supernatants (ASC) obtained from newly diagnosed ovarian cancer patients. Recombinant CR1 (rCRl) or recombinant CD46 (rCD46) were added to co-cultures to inhibit C3b mediated signaling via CR1. Supernatants from T cell - ASC co-cultures were obtained 96hrs post activation and employed in an IL-2 ELISA. Symbols represent individual ascites samples.

[0069] Example 3

[0070] Example 3 is illustrated by FIG. 3, which provides data demonstrating that CD35 and CD46 are expressed by T cells and expression profile is not altered by stimulation, exposure to ASC or with treatment using a complement inhibitor. To obtain the data shown in this Example, T cells were isolated from normal donor peripheral blood, stimulated with CD3 / CD28 Dynabeads and exposed to cryopreserved ascites supernatants (ASC) obtained from newly diagnosed ovarian cancer patients. Ascites were pre-treated with compstatin (Cp40) to inhibit complement activation or scrambled peptide (SCR) as controls. T cells were collected 24hr post activation, stained and used for flow cytometric analysis. Mean fluorescence intensities (MFIs) for CD8+ T cells represented here; similar results were observed for CD4+ T cells. Symbols represent individual ascites samples. Example 4

[0071] Example 4 is illustrated by FIGs. 4A-4B, which provides data demonstrating cell surface deposition of C3b / iC3b is increased on exposure to ASC and is significantly diminished by complement inhibition. To obtain the data shown in this Example, T cells were isolated from normal donor peripheral blood, stimulated with CD3 / CD28 Dynabeads and exposed to cryopreserved ascites supernatants (ASC) obtained from newly diagnosed ovarian cancer patients. Ascites were pre-treated with (A) compstatin (APL-38; provided by Apellis Pharmaceuticals)) to inhibit complement activation or scrambled peptide (SCR) as controls or (B) recombinant CR1 (rCRl) to inhibit C3b mediated signaling via CR1. T cells were collected 5hrs post activation, stained and used for flow cytometric analysis. Percentage CD8+ T cells positive for C3b / iC3b deposition on the cell surface represented here; similar results were observed for CD4+ T cells. Symbols represent individual ascites samples. “rCD35” means rCR-1.

[0072] Example 5

[0073] This Example is illustrated by FIGs. 5A-5E. As described in the description of FIGs. 5A-5E above, small molecule inhibitors of C3aR, C5aR, and cathepsin L (mediating intracellular C3 activation) and anti-C7 had no significant effect on T cell cytokine responses (not shown). C3b / iC3b deposition on T cells was increased with ASC exposure and reduced to background levels by C3 inhibition and by addition of recombinant complement receptor-1 (rCR-1; CD35). The data provide additional validation of the function of rCR-1 which fully abrogated the suppressive effect of ASC on T cells.

Claims

Claims:

1. A method for treatment of cancer, the method comprising administering to an individual in need of treatment a recombinant complement receptor- 1 (rCR-1).

2. The method of claim 1, wherein the rCR-1 inhibits binding of C3b to membranebound CR-1.

3. The method of claim 1, wherein the rCR-1 is administered as a component of a pharmaceutical formulation.

4. The method of claim 1, wherein rCR-1 expressed by a cell that is configured to also express a chimeric antigen receptor (CAR) or a bi-specific binding partner.

5. The method of claim 4, wherein the rCR-1, and the CAR or the bi-specific binding partner are expressed by a leukocyte that is optionally a T cell.

6. The method of claim 5, wherein the bi-specific binding partner is in the form of a Bi- specific T cell engager (BiTE) and wherein optionally engagement of the BiTE with a cancer cell targeted by the BiTE promotes secretion of the rCR-1.

7. The method of claim 5, wherein the cell is configured to express the CAR, and wherein optionally engagement of the CAR promotes secretion of the rCR-1.

8. The method of any one of claims 1-7, wherein the cancer is ovarian cancer.

9. A modified leukocyte that is configured to express a chimeric antigen receptor (CAR) and rCR-1.

10. A modified leukocyte that is configured to express a Bi-specific T cell engager (BiTE) and rCR-1.

11. A method comprising selecting an individual who has been diagnosed with cancer, the method comprising administering to the individual a pharmaceutical composition comprising recombinant complement receptor-1 (rCR-1) or a polynucleotide encoding the rCR-1, or cells that produce the rCR-1.

12. The method of claim 11, wherein the individual has been diagnosed with ovarian cancer.

Images