Chimeric antigen receptors and CXCR5-binding CAR-T cells

Abstract

The present invention relates to a separated chimeric antigen receptor polypeptide (CAR), wherein the CAR comprises an extracellular antigen binding domain, and the extracellular antigen binding domain comprises an antibody or antibody fragment that binds to a human CXC chemokine receptor type 5 (CXCR5) protein. The present invention further relates to nucleic acid molecules encoding the CAR of the present invention, genetically modified immune cells expressing the CAR of the present invention, preferably T cells, and the use of the cells in the treatment of medical conditions related to the presence of pathogenic cells expressing CXCR5, wherein the pathogenic cells expressing CXCR5 are preferably pathogenic mature B cells and / or memory B cells and / or pathogenic T cells and / or follicular helper T cells, particularly mature B cell non-Hodgkin lymphoma (B-NHL), T cell non-Hodgkin lymphoma, or autoantibody-dependent autoimmune diseases, preferably selected from systemic lupus erythematosus (SLE) or rheumatoid arthritis.

Description

[0001] describe

[0002] The present invention relates to a separated chimeric antigen receptor polypeptide (CAR), wherein the CAR comprises an extracellular antigen binding domain, which comprises an antibody or antibody fragment that binds to a CXC chemokine receptor type 5 (CXC chemokine receptor type 5, CXCR5) protein. The present invention further relates to nucleic acid molecules encoding the CAR of the present invention, genetically modified immune cells expressing the CAR of the present invention, preferably T cells, and the use of the cells in the treatment of medical conditions associated with the presence of pathogenic cells expressing CXCR5, wherein the pathogenic cells expressing CXCR5 are preferably pathogenic mature B cells and / or memory B cells and / or pathogenic T cells and / or follicular helper T cells, particularly mature B cell non-Hodgkin lymphoma (B-NHL), T cell non-Hodgkin lymphoma, or autoantibody-dependent autoimmune diseases, wherein the autoantibody-dependent autoimmune diseases are preferably selected from systemic lupus erythematosus (SLE) or rheumatoid arthritis. Background Art

[0003] B-NHL is heterogeneous and can be distinguished by aggressive versus indolent course. The standard of care is typically a combination of antibody / chemotherapy, either alone or in combination with autologous stem cell transplantation, immunomodulatory drugs, radiation, proteasome inhibitors, signaling pathway inhibitors, and, in rare cases, allogeneic stem cell transplantation. Because the median age at diagnosis is >66 to 72 years in many B-NHL entities, comorbidities exist that preclude intensive and prolonged chemotherapy or even allogeneic bone marrow transplantation.

[0004] In mature B-cell lymphomas, inhibitors of B-cell receptor (BCR) signaling, most importantly ibrutinib, have led to dramatic advances in response rates. Despite the initial high sensitivity to this class of kinase inhibitors, it is uncertain whether tumor eradication can be achieved; secondly, several studies have shown that the evolution of clonal lymphomas and leukemias leads to resistance to Bruton’s tyrosine kinase (BTK) inhibition. Therefore, the rapid emergence of secondary resistance to targeted therapies indicates an urgent need to find tolerable salvage therapy options, especially for patients who have been treated with several other chemotherapies and have a reduced clinical picture (IPI score).

[0005] Adoptive chimeric antigen receptor (CAR)-T cell therapy targeting the CD19 antigen widely expressed on leukemia and lymphoma B cells has brought substantial clinical efficacy. Currently, more than 40 CD19 CAR-T cell studies have been registered by the FDA for the treatment of B-NHL and B-ALL (www-clinicaltrials.gov). However, in anti-CD19 antibody or CAR-T cell therapy directly targeting B-NHL, resistance is generated due to antigen loss. Recent studies have shown that after anti-CD19 CAR-T cell treatment, escape variants caused by the selection of alternative splicing CD19 isoforms appear, thereby losing the relevant CD19 CAR epitope.

[0006] Therefore, CXCR5 becomes an alternative target for immunotherapy of B-cell lymphoma in addition to existing therapeutic monoclonal antibodies or CAR-T cell therapies.

[0007] B-cell-derived lymphoproliferative diseases with prominent nodal lodging, such as acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), frequently express the homeostatic chemokine receptor CXCR5. CXCR5 is physiologically expressed on mature circulating B cells and a small fraction of CD4+ T cells (follicular helper T cells (Tfh)), and regulates their homeostatic trafficking and homing to B cell follicles within secondary lymphoid organs. Importantly, CXCR5 is not expressed on B cell precursors within the bone marrow (BM), nor do plasma cells express this receptor.

[0008] To the best of the inventors' knowledge, no alternative anti-CXCR5 CAR constructs have been previously described and no anti-CXCR5 antibody studies are currently underway in connection with the medical methods of the invention.

[0009] Panjideh et al. (International Journal of Cancer, Vol. 135, No. 11, April 29, 2014) describes the use of CXCR5 bispecific antibodies in the treatment of non-Hodgkin's lymphoma. Sadelain et al. (Cancer Discovery, Vol. 3, No. 4, April 1, 2013) reviewed various CAR technologies, but did not mention CXCR5. WO 2016 / 090034 discloses multiple possible targets for CAR constructs. CD185 (CXCR5) is mentioned, but there are no details about CAR components, medical uses, or no mention of target relevance at all. WO2016 / 164731 describes the use of CAR-T cells against multiple B cell target antigens. There is no mention of CXCR5 as a CAR T target.

[0010] Alternative therapies for medical conditions such as those listed above are under development or currently established, such as anti-CD19 CAR constructs, standard therapies such as cytotoxic chemotherapy, corticosteroids, immunomodulators (such as IMIDs), proteasome inhibitors, autologous stem cell transplantation, allogeneic stem cell transplantation, signal transduction inhibitors, and antibodies rituximab against CD20 and oletumomab against CD19, as well as a bispecific antibody (BITE) comprising a Fab fragment targeting CD19 and an anti-CD3 fragment (blinatumomab).

[0011] Although many potential alternative therapies are under development for diseases targeting pathogenic B and T cells, there remains a strong need to provide effective means of addressing such medical conditions. Summary of the invention

[0012] In view of the prior art, the technical problem underlying the present invention is to provide a medicament suitable for treating diseases associated with pathogenic B cells and / or T cells, in particular non-Hodgkin's lymphoma or autoantibody-dependent autoimmune diseases.

[0013] This problem is solved by the features of the independent claim. Preferred embodiments of the invention are provided by the dependent claims.

[0014] Therefore, the present invention relates to a chimeric antigen receptor polypeptide (CAR), comprising:

[0015] i. an extracellular antigen binding domain comprising an antibody or antibody fragment that binds to a CXC chemokine receptor type 5 (CXCR5) protein,

[0016] ii. transmembrane domain, and

[0017] iii. Intracellular domain.

[0018] Therefore, the present invention relates to anti-CXCR5 CAR constructs and corresponding immune cells expressing the constructs, preferably retaining normal hematopoietic cells such as T cells (except Tfh or other T cell lymphomas described herein), plasma B cells and CAR-T cell products of their bone marrow precursors while conferring high cytotoxic activity to human T cells against determined mature B-NHL. In a preferred embodiment, all myeloid cells and NK cells are also retained; because the CAR-T cell products of the present invention have no activity on these cells.

[0019] In a preferred embodiment of the immunotherapy of the present invention, the patient-derived T cells are preferably transduced with retroviral to express an artificial immune receptor as described herein, which consists of an extracellular antibody-derived antigen recognition portion fused to a transmembrane portion; followed by an intracellular signaling domain. Thus, the constructs described herein confer anti-tumor cytolytic ability to the transduced T cells.

[0020] Due to the preferred autologous transfer of T cells, graft-versus-host disease cannot occur when treated with the CAR-T of the present invention. Memory T cell formation can be developed, which is important for preventing relapse.

[0021] Such mature B-NHL entities include, but are not limited to, certain stages of follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia.

[0022] For the first time, anti-CXCR5 CAR-T cells will be able to target tumor cells within the tumor microenvironment, as Tfh cells that promote lymphoma growth will be eradicated simultaneously. Tumor cells within a given tumor entity are all positive for the target antigen CXCR5, thus excluding unwanted positive selection of low-expressing / non-expressing tumor cells.

[0023] In a preferred embodiment, the anti-CXCR5 CAR-T cells described herein are suitable for treating mature B-NHL patients who are not suitable for other therapies. More specifically, embodiments of the present invention relate to the treatment of the following patient groups:

[0024] i) Patients with multidrug resistance,

[0025] ii) Patients who are not eligible for allogeneic stem cell transplantation,

[0026] iii) patients with comorbidities that preclude further chemotherapy,

[0027] iv) Elderly patients who cannot tolerate chemotherapy,

[0028] v) CAR is indicated for salvage therapy even after progressive disease and failure of multiple other standard-of-care therapies,

[0029] vi) is applicable even in situations where the antigen density on the target tumor cells is low, where the antibody may fail, and / or

[0030] vii) Suitable as a monotherapy which is not the case for antibodies.

[0031] The anti-CXCR5 CAR described herein confers the high affinity necessary for T cell anti-tumor efficacy. It has been demonstrated that the anti-CXCR5 CAR of the present invention does not confer T cell reactivity to physiological plasma B cells, T cells (except Tfh cells and T cells expressing specific pathological CXCR5), NK cells, all myeloid cell lineages and their precursors. Therefore, the present invention has unprecedented low off-target reactivity to other hematopoietic tissues.

[0032] In contrast to anti-CD19 CAR-T cells, the anti-CXCR5 CARs of the present invention have no unwanted reactivity to immature B-NHL, precursor B-cell neoplasia, or physiologically benign B-cell precursors.

[0033] As shown in the following examples, anti-CXCR5 CAR-T cells were activated when exposed to a human B-NHL tumor cell line expressing CXCR5 in an in vitro co-culture system. These T cells then developed an effector phenotype with high levels of IFN-γ secretion, which predicts cytotoxic activity.

[0034] Additionally, cytotoxicity assays (51Cr release) against selected target cell lines, B-NHL, B and T cell leukemias, CXCR5 negative cells and CXCR5 transfectants demonstrated that selective cytotoxicity was only achieved in cell lines positive for CXCR5.

[0035] Additional preclinical testing includes: i) in vitro cytotoxicity testing on appropriate patient-derived B-NHL cell lines and ii) in vivo testing of anti-CXCR5 CAR activity on xenografted B-NHL cell lines.

[0036] Therefore, the CAR of the present invention represents a surprising and beneficial method for treating the medical conditions described herein. Anti-CXCR5 CAR has not been tried or described as a promising method for treating NHL before. Due to the selectivity of the marker, minimal (if not absent) undesirable side effects also represent beneficial and surprising aspects of the present invention. Especially in patients who have developed resistance to anti-CD19 treatment, the present invention represents a very promising method for eliminating malignant tumors.

[0037] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, wherein when the CAR is expressed in a genetically modified immune cell, preferably a T lymphocyte, the immune cell binds to CXCR5 on the surface of a cell expressing CXCR5 and becomes activated, thereby inducing cytotoxic activity against the cell expressing CXCR5.

[0038] Examples of cells expressing CXCR5 are known to the skilled person and can be identified by further screening of cancer or other pathogenic cells. Cell lines expressing CXCR5 are preferably DOHH-2, OCI-Ly7, SU-DHL4, JeKo-1, JVM-3, MEC-1 and / or SC-1.

[0039] In one embodiment, the present invention relates to a chimeric antigen receptor polypeptide (CAR) comprising:

[0040] - an extracellular antigen binding domain comprising an antibody or antibody fragment that binds to a CXC chemokine receptor type 5 (CXCR5) protein, wherein the antibody or antibody fragment comprises the VH and VL domains of a single-chain antibody fragment, wherein preferably a linker polypeptide is located between the VH and VL domains, wherein the linker is preferably configured not to interfere with the antibody fragment-CXCR5 antigen interaction;

[0041] - a spacer polypeptide (also called a hinge) located between the extracellular antigen binding domain and the transmembrane domain, wherein the spacer polypeptide is preferably configured so as not to interfere with antibody fragment-CXCR5 antigen interaction and / or T cell activation when the CAR is expressed in a T cell expressing the CAR;

[0042] - a transmembrane domain, wherein the transmembrane domain is preferably configured so as not to interfere with antibody fragment-CXCR5 antigen interaction and / or T cell activation when the CAR is expressed in a T cell expressing the CAR;

[0043] - and an intracellular domain, wherein the intracellular domain comprises a co-stimulatory domain and a signaling domain, wherein the intracellular domain is preferably configured to provide a signal to stimulate T cell activation when binding to the CXCR5 target, for example by increasing cytokine production and / or promoting T cell replication, thereby resulting in a cytotoxic effect.

[0044] Therefore, the CAR of the present invention can take various forms, including possible different protein sequences for each functional domain described herein. Technicians can select and test the desired function of CAR, for example, based on the experimental methods demonstrated in the following examples. Therefore, those skilled in the art can use conventional methods to evaluate the functional efficacy of any given specific protein sequence to be used for the CAR of the present invention in any functional domain discussed herein. For example, various joint polypeptide sequences between VH and VL domains, various spacer polypeptide sequences (also referred to as hinges) between extracellular antigen binding domains and transmembrane domains, various transmembrane domains, and various intracellular domains preferably comprising costimulatory domains and signaling domains can be used.

[0045] In an embodiment of the present invention, CAR and each element or domain mentioned herein are constructed so as not to deleteriously interfere with antibody fragment-CXCR5 antigen interaction, not to deleteriously interfere with T cell activation when the CAR is expressed in a T cell expressing the CAR, and not to deleteriously interfere with CAR providing a signal to stimulate T cell activation when bound to the CXCR5 target.

[0046] Experimental methods for evaluating these properties of CXCR5 CAR are described herein, so that the present invention is considered to cover various functional sequence variants and combinations of domains of the type described herein, without being limited to the specific sequences disclosed by the following examples. For example, specific activation of CAR-T cells of the present invention by tumor cells expressing CXCR5 can be demonstrated by the release of IFN-γ, IL-2 and TNF-α, as shown below.

[0047] To the best of the inventors' knowledge, the present invention relates to the first described CXCR5 CAR, and the first functional evidence of the desired therapeutic effect of CXCR5 CAR in a medical setting. Independent of the specific sequences employed in the various functional domains described herein, the provision of CXCR5 CAR alone represents a significant and beneficial breakthrough in the treatment of many diseases associated with pathogenic mature B cells and / or memory B cells and / or pathogenic T cells and / or follicular helper T cells.

[0048] Embodiments related to the antigen binding domain of CAR:

[0049] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, wherein the antigen binding domain comprises a variable heavy chain (VH), wherein the VH comprises:

[0050] - a heavy chain complementarity determining region 1 (H-CDR1) having at least 80% sequence identity to SEQ ID NO 1 (GFTFSTSG),

[0051] - a heavy chain complementarity determining region 2 (H-CDR2) having at least 80% sequence identity to SEQ ID NO 2 (ISSSSGFV), and

[0052] - a heavy chain complementarity determining region 3 (H-CDR3) having at least 80% sequence identity to SEQ ID NO 3 (ARSEAAF),

[0053] and a variable light chain (VL), the VL comprising:

[0054] - a light chain complementary determining region 1 (L-CDR1) having at least 80% sequence identity to SEQ ID NO 4 (KSRLSRMGITP),

[0055] - a light chain complementarity determining region 2 (L-CDR2) having at least 66% sequence identity to SEQ ID NO 5 (RMS), and

[0056] - a light chain complementarity determining region 3 (L-CDR3) having at least 80% sequence identity to SEQ ID NO 6 (AQFLEYPPT).

[0057] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, comprising: a VH domain comprising the CDR sequences of SEQ ID NO.1, SEQ ID No.2 and SEQ ID NO.3; and a VL domain comprising the CDR sequences of SEQ ID NO.4, SEQ ID No.5 and SEQ ID NO.6.

[0058] In a preferred embodiment, sequence variants having 80% or higher sequence identity with the specific CDR sequences of SEQ ID NOs 1 to 6 retain binding to CXCR5 and have substantially the same or similar functional properties as the VH and VL domains having the specific CDR sequences of SEQ ID NOs 1 to 6, i.e., substantially the same or similar CXCR5 binding in terms of affinity, specificity and epitope binding pattern.

[0059] The amino acid sequence of the scFV fragment was originally obtained from a rat anti-human CXCR5 antibody and was modified in various improvements, such as by humanization of the VL and VH chains, to allow folding and expression in the context of a transmembrane receptor structure.

[0060] Furthermore, the order of the light and heavy chain fragments can be reversed depending on the desired configuration of the antigen-binding fragment.

[0061] Additionally, in some embodiments, the linker sequence between the heavy and light chains has been modified, e.g., by shortening, to enhance CAR function.

[0062] In addition, the nucleic acid sequence encoding CAR has been codon optimized to improve the expression of CAR.

[0063] These modifications enable adequate surface expression on T cells and still maintain appropriate antigen binding. High affinity and high avidity enable CAR-T cells to i) recognize tumor target cells with high, medium or low CXCR5 surface expression, ii) activate against tumor target cells with high, medium or low CXCR5 surface expression, and iii) kill tumor target cells with high, medium or low CXCR5 surface expression.

[0064] To the best of the inventors' knowledge, neither anti-CXCR5 CARs nor humanized anti-CXCR5 antibodies have been previously described in the art.

[0065] Due to the high affinity and avidity of the antigen binding domain of the anti-CXCR5 CAR-T cells described herein, even mature B-NHL with low expression of CXCR5 can be recognized, allowing T cell activation and tumor cell killing.

[0066] The anti-CXCR5 CAR-T cell products described herein are characterized by unique properties.

[0067] Anti-CXCR5 CAR as described herein has high affinity and confers high specificity and avidity to T cells. These properties enable CAR-T cells to i) recognize tumor target cells with high and low CXCR5 surface expression, ii) activate against tumor target cells with high and low CXCR5 surface expression, and iii) kill tumor target cells with high and low CXCR5 surface expression.

[0068] The amount of CXCR5 antigen expressed on the surface of tumor cells can be quantified by using anti-CXCR5 antibodies coupled to fluorescent dyes in combination with Quantibrite beads (from Becton Dicksinson). The preferred method for quantifying CXCR5 antigen expressed on the surface of tumor cells is "fluorescence activated cell sorting / cell analysis" (FACS). The fluorescence intensity of the beads is accurately correlated to the amount of fluorescent antibodies bound to the cells, which is a measure of the number of CXCR5 molecules on the cells.

[0069] The VH and VL fragments described herein can be arranged in a variety of configurations in CAR, and still maintain high specificity and high affinity for the target epitope. In some embodiments, CAR can be constructed in a VH-VL or VL-VH configuration, changing in a joint, hinge, transmembrane domain, co-stimulatory domain, and / or activation domain, and still maintain its efficacy. This surprising feature of the present invention allows the design of CARs directly targeting CXCR5 to have greater flexibility, so that if any further development is necessary or desired, the CAR structure can be further modified and / or optimized based on the VH and VL domains described herein.

[0070] Sequence alignment of rat and humanized sequences is Figure 4 . Therefore, the following sequences also include universal sequences representing rat and humanized forms of antigen-binding domains. In the following sequences, each X represents a potential amino acid change. Preferred amino acid substitutions are those described for each potential position of the change.

[0071] The present invention encompasses all possible combinations of potential modifications of any given potential variant residues proposed herein (as indicated by X in the "universal sequence"). By combining one or more of these substitutions, humanized variants exhibiting the desired binding properties of the rat antigen binding domains demonstrated herein can be produced. CARs described herein or portions thereof also include sequences having at least 70%, 80%, preferably 90% sequence identity to humanized sequences explicitly disclosed or disclosed by sequence formula.

[0072] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, comprising:

[0073] A VH domain having at least 70%, 80% sequence identity, preferably at least 85%, 90%, 95% or 100% sequence identity to SEQ ID NO 7 as follows:

[0074] EVQLVESGGGLVQPGX1SLX2LSCX3ASGFTFSTSGMX4WFRQAPGKGLX5WVX6YISSSSGFVYADX7VKGRFTISRDNAQNX8LYLQX9NSLX10X11EDTAX12YYCARSEAAFWGQGTLVTVSS,

[0075] Wherein X1 to X12 can be any amino acid, preferably X1: G or K, X2: R or K; X3: A or S; X4: N or H; X5: E or D; X6: S or A; X7: S or A; X8: S or T; X9: M or L; X10: R or K, X11: A or S; X12: V or I;

[0076] and a VL domain having at least 80% sequence identity, preferably at least 85%, 90%, 95% or 100% sequence identity to SEQ ID NO 8 as follows:

[0077] DIVLTQX1PRSX2PVTPGEX3ASISCRSX4KSRLSRMGITPLNWYLQKPGX5SPQLLIYRMSNX6ASGVPDRFSGSGSX7TDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK,

[0078] Wherein X1 to X7 can be any amino acid, preferably X1: S or A; X2: L or V; X3: P or S; X4: S or N; X5: Q or K; X6: R or L; X7: G or E.

[0079] In a preferred embodiment, sequence variants having 80% or greater sequence identity to the specific VH and VL sequences listed herein retain binding to CXCR5 and have substantially the same or similar functional properties as the VH and VL domains having the specific sequences listed herein, i.e., substantially the same or similar CXCR5 binding in terms of affinity, specificity, and epitope binding pattern.

[0080] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, comprising:

[0081] - VH domain according to SEQ ID NO 9

[0082] (EVQLVESGGGLVQPGGSLRLSCAASGFTFSTSGMNWFRQAPGKGLEWVSYISSSSGFVYADSVKGRFTISRDNAQNSLYLQMNSLRAEDTAVYYCARSEAAFWGQGTLVTVSS)

[0083] - or according to SEQ ID NO 10

[0084] (EVQLVESGGGLVQPGKSLKLSCSASGFTFSTSGMHWFRQAPGKGLDWVAYISSSSGFVYADAVKGRFTISRDNAQNTLYLQLNSLKSEDTAIYYCARSEAAFWGQGTLVTVSS);

[0085] - and a VH domain according to SEQ ID NO 11

[0086] (DIVLTQSPRSLPVTPGEPASISCRSSKSRLSRMGITPLNWYLQKPGQSPQLLIYRMSNRASGVPDRFSGSGSGTDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK)

[0087] - or according to SEQ ID NO 12 as follows

[0088] (DIVLTQAPRSVSVTPGESASISCRSNKSRLSRMGITPLNWYLQKPGKSPQLLIYRMSNLASGVPDRFSGSGSETDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK).

[0089] In other embodiments, the present invention relates to chimeric antigen receptor (CAR) polypeptides, which include one or more joints, spacers, transmembrane domains and signaling domains. In one embodiment, CAR includes an intracellular domain, which includes a costimulatory domain and a signaling (activation) domain.

[0090] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, wherein the extracellular antigen binding domain comprises a linker polypeptide located between the VH and VL domains, wherein the linker is preferably selected from:

[0091] - Whitlow (SEQ ID NO 13; GSTSGSGKPGSGEGSTKG), or

[0092] -Gly-Ser (SEQ ID NO 14; SSGGGGSGGGGSGGGGS) linker, or

[0093] - a linker having a sequence identity of at least 80% to SEQ ID NO 13 or 14.

[0094] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, further comprising a spacer polypeptide located between the extracellular antigen binding domain and the transmembrane domain, wherein the spacer is selected from:

[0095] - IgG1 spacer (SEQ ID NO 15:

[0096] PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK),

[0097] -IgG1Δ spacer (SEQ ID NO 16:

[0098] PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSSLSPGKK),

[0099] -IgG4(Hi-CH2-CH3) spacer (SEQ ID NO 17:

[0100] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK),

[0101] -IgG4(Hi-CH3) spacer (SEQ ID NO 18:

[0102] ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK),

[0103] - IgG4 (Hi) spacer (SEQ ID NO 19; ESKYGPPCPPCP) or

[0104] - a spacer having a sequence identity of at least 80% to any one of SEQ ID NOs 15 to 19.

[0105] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, wherein the transmembrane domain is selected from:

[0106] - CD8 alpha domain (SEQ ID NO 20; IYIWAPLAGTCGVLLLSLVITLYC), or

[0107] - CD28 domain (SEQ ID NO 21; FWVLVVVGGVLACYSLLVTVAFIIFWV), or

[0108] - a transmembrane domain having at least 80% sequence identity to SEQ ID NO 20 or 21.

[0109] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, wherein the intracellular domain comprises:

[0110] - a co-stimulatory domain selected from the group consisting of a 4-1BB co-stimulatory domain (SEQ ID NO 22:

[0111] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), and / or

[0112] -CD28 co-stimulatory domain (SEQ ID NO 23:

[0113] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSL), or

[0114] - a co-stimulatory domain comprising both 4-1BB (SEQ ID NO 22) and a CD28 co-stimulatory domain (SEQ ID NO 23) arranged adjacently, or

[0115] - a co-stimulatory domain having at least 80% sequence identity to SEQ ID NO 22 or 23.

[0116] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, which further comprises a signaling domain (also called an activation domain), wherein the signaling domain is

[0117] - CD3ζ (4-1BB or CD28) signaling domain (SEQ ID NO 24; LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), or

[0118] - a signalling domain having at least 80% sequence identity to SEQ ID NO 24.

[0119] In one embodiment, the present invention relates to a chimeric antigen receptor (CAR) polypeptide as described herein, comprising a sequence according to any one of SEQ ID NOs 25, 26, 27, 28 or 29.

[0120] The exchange of signaling domains meets the needs of strong and rapid effector phase (CD28 costimulatory domain) or long-term relapse control (4-1BB signaling domain) guaranteed by T cell memory population. As demonstrated herein, various signaling domains can be exchanged in a variety of configurations, thereby providing flexibility to CAR in its design without losing favorable binding properties.

[0121] In other embodiments of the present invention, CAR may include the following configurations:

[0122] -H28:MP71-hCXCR5-VH-Whitlow-VL-IgG1-CD28-CD28-CD3z

[0123] -R28:MP71-ratCXCR5-VH-Whitlow-VL-IgG1-CD28-CD28-CD3z

[0124] -HBB1:MP71-hCXCR5-VH-Whitlow-VL-IgG1Δ-CD8α-4-1BB-CD3z

[0125] -HBB2:MP71-hCXCR5-VH-Whitlow-VL-IgG1-CD28-4-1BB-CD3z

[0126] -H28BB:MP71-hCXCR5-VH-Whitlow-VL-IgG1-CD28-CD28-4-1BB-CD3z

[0127] The specific configuration is intended as a preferred but non-limiting embodiment. The above configuration is not intended to be limited by the specific sequence of those embodiments described herein. In the case of these configurations, sequence changes are possible and are covered by the scope of the present invention.

[0128] As variants are used for linkers, spacers, transmembrane domains and intracellular domains (by adding alternative components), it is apparent that one skilled in the art can exchange the various components as desired and still maintain the CXCR5 binding properties and thus the desired biological effect.

[0129] In a preferred embodiment, in combination with MP71-vector and γ-retroviral expression system, an abnormally high transduction rate of human T cells can be achieved. The transduction system is variable due to the modular design of the CAR construct, which means that both lentivirus and transposon can be used, depending on the needs and preferences of the technicians when implementing the present invention. The transfer of genetic information / nucleic acid molecules for CXCR5 CAR also includes CrispR / Cas and TALEN-mediated insertion into target cell lines, preferably T lymphocytes, natural killer cells and induced pluripotent stem cells iPS. The present invention covers all suitable methods for transferring the genetic information / nucleic acid molecules of CXCR5 CAR to cells expressing the CAR, and those skilled in the art can select suitable methods when implementing the present invention. For example, a variety of methods for transforming T cells are known in the art, including any given virus-based gene transfer method (e.g., based on modified retroviridae) and non-viral methods (e.g., DNA-based transposons and methods for directly transferring mRNA by electroporation).

[0130] Additionally, the signaling components of the CAR constructs have been exchanged in a three-step cloning procedure that allows for the combination of components of a clinically applicable anti-CXCR5 CAR and customized construction by skilled artisans.

[0131] In another aspect of the present invention, the present invention relates to an isolated nucleic acid molecule, which is preferably in the form of a vector, such as a viral vector or a transposon vector, preferably a Sleeping Beauty vector, wherein the isolated nucleic acid molecule is selected from the group consisting of:

[0132] a) a nucleic acid molecule comprising the following nucleotide sequence

[0133] - the nucleotide sequence encodes a chimeric antigen receptor (CAR) polypeptide according to any embodiment of the CAR described herein,

[0134] - the nucleotide sequence encodes an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain, wherein the extracellular antigen-binding domain is encoded by at least one sequence of SEQ ID NO: 37 or 38 and at least one sequence of SEQ ID NO: 39 or 40,

[0135] - the nucleotide sequence is according to SEQ ID No. 31, 32, 33, 34 or 35, and / or

[0136] b) a nucleic acid molecule complementary to the nucleotide sequence according to a);

[0137] c) a nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity to be functionally similar / equivalent to the nucleotide sequence according to a) or b), preferably comprising a nucleotide sequence having a sequence identity of at least 80% to the nucleotide sequence according to a) or b);

[0138] d) a nucleic acid molecule which, due to the genetic code, is degenerate to a nucleotide sequence according to a) to c); and / or

[0139] e) A nucleic acid molecule of a nucleotide sequence according to a) to d), which is modified by deletion, addition, substitution, translocation, inversion and / or insertion and is functionally similar / equivalent to the nucleotide sequence according to a) to d).

[0140] In a preferred embodiment of the present invention, the isolated nucleic acid molecule is preferably in the form of a vector, such as a viral vector or a transposon vector, preferably a Sleeping Beauty vector, and the isolated nucleic acid molecule is selected from the group consisting of:

[0141] a) a nucleic acid molecule comprising the following nucleotide sequence

[0142] - the nucleotide sequence encodes a chimeric antigen receptor (CAR) polypeptide according to any embodiment of the CAR described herein,

[0143] - the nucleotide sequence encodes an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain, wherein the extracellular antigen-binding domain is encoded by at least one sequence of SEQ ID NO: 37, 53 or 38 and at least one sequence of SEQ ID NO: 39, 54 or 40,

[0144] - the nucleotide sequence is according to SEQ ID No. 31, 32, 33, 34 or 35, and / or

[0145] b) a nucleic acid molecule complementary to the nucleotide sequence according to a);

[0146] c) a nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity to be functionally similar / equivalent to the nucleotide sequence according to a) or b), comprising a nucleotide sequence having a sequence identity of at least 80%, preferably 90% or 95% to the nucleotide sequence according to a) or b), wherein the functional similarity relates to binding to the CXCR5 target antigen, and when the corresponding T cells express the construct, the CAR-T cell product confers cytotoxic activity of the T cells against mature B-NHL while retaining normal hematopoietic cells, such as T cells (except Tfh or other T cell lymphomas described herein), plasma B cells and their bone marrow precursors; and / or

[0147] d) Nucleic acid molecules which, due to the genetic code, have a nucleotide sequence according to a) to c).

[0148] The term degenerate is (or degenerates into) refers to the difference of the nucleotide sequence of the nucleic acid molecule, but according to the genetic code, the difference of the amino acid protein product of the nucleotide sequence will not result in translation. In one embodiment, the nucleic acid molecule relates to the above-mentioned molecules that meet a) and b), a), b) and c), or a), b) and d).

[0149] Preferred amino acid and nucleotide sequences of the present invention are:

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] Another aspect of the present invention relates to a vector comprising a nucleic acid molecule as described herein, the vector is preferably a viral vector, more preferably a γ-retroviral vector. In another aspect of the present invention, the present invention relates to a transposon vector encoding and preferably capable of expressing the CAR of the present invention, the transposon vector is preferably a Sleeping Beauty vector.

[0161] Another aspect of the present invention relates to a genetically modified immune cell comprising a nucleic acid molecule or vector as described herein, and / or expressing a CAR as described herein.

[0162] In a preferred embodiment, the "Sleeping Beauty" transposon system, in particular the Sleeping Beauty transposase, is used to genetically modify immune cells intended to be administered to treat the diseases mentioned herein with nucleic acids as described herein, which encode and express anti-CXCR5 CAR as described herein. In the context of the present invention, the Sleeping Beauty transposon system is a synthetic DNA transposon designed to introduce precisely defined DNA sequences into vertebrate chromosomes for the purpose of modifying immune cells to express CAR as described herein. The Sleeping Beauty transposon combines the advantages of viruses and naked DNA. Viruses have been evolutionarily selected based on their ability to infect and replicate in new host cells. At the same time, cells have evolved major molecular defense mechanisms to protect themselves from viral infection. It is also important to avoid the use of viruses for social and regulatory reasons. Therefore, the use of non-viral vectors, such as the Sleeping Beauty system, can avoid many but not all defenses of cells against vectors. For this reason, the Sleeping Beauty system enables particularly effective and safe genetic modification of immune cells for administration to patients.

[0163] In other embodiments of the present invention, CrispR / Cas and TALEN-mediated insertion of nucleic acids encoding CXCR5 CAR can be used. CrispR / Cas known to technicians to be adapted to naturally occurring processes in bacteria can be used to accurately and efficiently edit DNA to insert suitable coding sequences into immune cells of interest, preferably T cells. Cas9 is a protein that acts as a molecular pair of scissors and can be guided to a specific DNA sequence by a related RNA molecule (guide RNA). When Cas9 reaches its target position on DNA, it contributes to changes in the local genetic code, thereby affecting the function of the gene. CRISPR / Cas9 can deliver the CAR gene to a very specific site within the T cell genome, which can reduce the risk of gene insertion at an incorrect or undesirable position.

[0164] In one embodiment, the immune cell is preferably selected from T lymphocytes or NK cells, more preferably cytotoxic T lymphocytes.

[0165] In a preferred embodiment, the immune cell comprising a nucleic acid molecule or vector as described herein and / or expressing a genetically modified CAR as described herein is characterized in that the immune cell is a CD4+ and / or CD8+ T cell, preferably a mixture of CD4+ and / or CD8+ T cells. These T cell populations, and preferably compositions comprising CD4+ and CD8+ transformed cells, exhibit particularly effective cytolytic activity for various malignant B cells (eg, B-NHL), preferably for those cells and / or related medical conditions described herein.

[0166] In a preferred embodiment, the genetically modified immune cells comprising nucleic acid molecules or vectors as described herein and / or expressing CARs as described herein are CD4+ and CD8+ T cells, preferably at a ratio of CD4+ and CD8+ T cells of 1:10 to 10:1, more preferably 5:1 to 1:5, 2:1 to 1:2 or 1:1. Administration of CXCR5-directed modified CAR-T cells expressing CARs described herein at the mentioned ratios, preferably at a 1:1 CD4+ / CD8+ ratio, produces beneficial properties during the treatment of the diseases mentioned herein, such as these ratios leading to improved therapeutic responses and reduced toxicity.

[0167] Another surprising aspect of the present invention is the improved stability of the CAR disclosed herein. Under appropriate conditions, the CAR polypeptide can be easily stored for a long time without any loss of binding affinity.

[0168] Another aspect of the invention relates to a genetically modified immune cell as described herein for use in treating a medical condition associated with the presence of pathogenic cells expressing CXCR5.

[0169] In one embodiment, the medical condition to be treated is associated with the presence of pathogenic mature B cells and / or memory B cells.

[0170] In one embodiment, the medical condition to be treated is mature B-cell non-Hodgkin lymphoma (B-NHL).

[0171] In other embodiments, the medical condition to be treated is a lymphoproliferative disorder of B-cell origin, preferably selected from the group consisting of acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL).

[0172] In one embodiment, the medical condition to be treated is associated with the presence of pathogenic T cells and / or follicular helper T cells.

[0173] In one embodiment, the medical condition to be treated is T-cell non-Hodgkin's lymphoma with or without leukemic tumor cell dissemination.

[0174] In one embodiment, the medical condition to be treated is a lymphoproliferative disorder of T-cell origin, preferably selected from the group consisting of angioimmunoblastic T-cell lymphoma, cutaneous T-cell lymphoma and T-cell lymphoma with leukemic disseminated.

[0175] Another aspect of the present invention relates to a genetically modified immune cell expressing the CAR of the present invention as described herein, which is used as a drug for treating autoantibody-dependent autoimmune diseases.

[0176] In a preferred embodiment, the autoimmune disease is selected from systemic lupus erythematosus (SLE) or rheumatoid arthritis.

[0177] Only recently, CAR-T cells have also been discussed as a targeted approach to treat autoantibody-mediated diseases (Ellebrecht et al. (2016) Science 353: 179-184). The ability to target CXCR5 would inhibit the co-localization of autoreactive B cells and Tfh cells, which would be of great benefit for treating autoimmune diseases.

[0178] Mild forms of autoimmune diseases are often initially treated with nonsteroidal anti-inflammatory drugs (NSAIDs) or disease-modifying antirheumatic drugs (DMARDs). More severe forms of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), including organ dysfunction due to active disease, are often treated with steroids combined with potent immunosuppressants such as cyclophosphamide, a cytotoxic agent that targets circulating cells. Only recently, belimumab, an antibody that targets the cytokine BAFF, which is found at elevated levels in the serum of patients with autoimmune diseases, received U.S. Food and Drug Administration (FDA) approval for SLE.

[0179] However, only newly formed B cells rely on BAFF to survive in the body, while memory B cells and plasma cells are less sensitive to selective BAFF inhibition (Jacobi et al. (2010) Arthritis Rheum 62:201–210). For rheumatoid arthritis (RA), TNF inhibitors were the first biological agents to be licensed, followed by abatacept, rituximab, and tocilizumab: they inhibit key inflammatory pathways involved in joint inflammation and destruction, but this comes at the expense of an increased risk of infection due to relative immunosuppression (Chan et al. (2010) Nat Rev Immunol 10:301–316, Keyser (2011) Curr Rheumatol Rev 7:77–87).

[0180] Rituximab is a monoclonal antibody that depletes B cells from the circulation and is increasingly prescribed for the treatment of RA in particular and also for the treatment of granulomatous diseases with polyangiitis and other anti-neutrophil cytoplasmic antibody-associated vasculitides. However, rituximab is not without risk and has a similar risk of adverse events as cyclophosphamide (Shah et al. (2015) ImmunoTargets and Therapy 4:173-183). Therefore, more sophisticated and durable approaches that ensure targeting of autoreactive B cells and autoantibody responses are necessary.

[0181] The present invention further relates to methods of treating the medical conditions described herein, generally comprising administering a therapeutically effective amount of a CAR or an immune cell expressing the CAR to a patient in need of such treatment. DETAILED DESCRIPTION

[0182] Chimeric Antigen Receptor:

[0183] According to the present invention, chimeric antigen receptor polypeptide (CAR) includes an extracellular antigen binding domain, a transmembrane domain and an intracellular domain, and the extracellular antigen binding domain includes an antibody or antibody fragment that binds to a target antigen. CAR is usually described as comprising an extracellular domain (antigen binding domain) derived from an antibody and an intracellular domain comprising a signal transduction component derived from a T cell signaling protein.

[0184] In a preferred embodiment, the extracellular domain preferably comprises a variable region constructed as a single-chain variable fragment (scFv) from the heavy chain and light chain of an immunoglobulin. ScFv is preferably connected to a hinge region that provides flexibility and transduces signals by anchoring the transmembrane portion to the intracellular signaling domain. The transmembrane domain is preferably derived from CD8α or CD28. In the first generation of CAR, the signaling domain is composed of the ζ chain of the TCR complex. The term "generation" refers to the structure of the intracellular signaling domain. The second generation CAR is equipped with a single costimulatory domain derived from CD28 or 4-1BB. The third generation CAR already includes two costimulatory domains, such as CD28, 4-1BB, ICOS or OX40, CD3ζ. Preferably, the present invention relates to second or third generation CAR.

[0185] In various embodiments, there is provided a genetically engineered receptor for redirecting the cytotoxicity of immune effector cells to B cells. These genetically engineered receptors are referred to herein as chimeric antigen receptors (CAR). CAR is a molecule that combines the specificity based on antibodies for a desired antigen (e.g., CXCR5) with the intracellular domain of the activated T cell receptor to produce a chimeric protein showing specific anti-CXCR5 cellular immune activity. As used herein, the term "chimeric" describes the composition of parts of different proteins or DNA from different sources.

[0186] The CAR considered herein comprises an extracellular domain (also referred to as a binding domain or antigen binding domain) bound to CXCR5, a transmembrane domain, and an intracellular domain or intracellular signaling domain. The anti-CXCR5 antigen binding domain of CAR is engaged with CXCR5 on the target cell surface to cause CAR aggregation, and the activation stimulus is delivered to the cell containing CAR. The main feature of CAR is the ability of CAR to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis, or the generation of molecules that mediate cell death of target antigen-expressing cells in a major histocompatibility complex (MHC)-independent manner, thereby utilizing the cell-specific targeting ability of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

[0187] In various embodiments, CAR comprises: an extracellular binding domain comprising a humanized CXCR5-specific binding domain; a transmembrane domain; one or more intracellular signaling domains. In a particular embodiment, CAR comprises: an extracellular binding domain comprising a humanized anti-CXCR5 antigen binding fragment thereof; one or more spacer regions; a transmembrane domain; one or more intracellular signaling domains.

[0188] "Extracellular antigen binding domain" or "extracellular binding domain" are used interchangeably and provide CAR with the ability to specifically bind to the target antigen CXCR5. The binding domain can be derived from natural, synthetic, semisynthetic or recombinant sources. Preferred is the scFv domain.

[0189] "Specific binding" should be understood to be understood by those skilled in the art, whereby the technician is well aware of the various experimental procedures that can be used to test binding and binding specificity. Methods for determining equilibrium association or equilibrium dissociation constants are known in the art. In many protein-protein interactions, some cross-reactions or background binding may be inevitable; this does not detract from the "specificity" of the binding between CAR and the epitope. "Specific binding" describes that an anti-CXCR5 antibody or its antigen-binding fragment (or a CAR comprising its antigen-binding fragment) binds to CXCR5 with a greater binding affinity than background binding. When the term "specificity" is considered in the understanding of the interaction between an antibody and an epitope, the term "directly directed to" also applies.

[0190] "Antigen (Ag)" refers to a compound, composition or substance that can stimulate the production of antibodies or T cell responses in an animal. In particular embodiments, the target antigen is an epitope of a CXCR5 polypeptide. "Epitope" refers to the region of an antigen to which a binding agent binds. An epitope can be formed by contiguous or discontinuous amino acids juxtaposed by the tertiary folding of a protein.

[0191] "Single-chain Fv" or "scFv" antibody fragments contain the VH and VL domains of an antibody, wherein these domains are present as a single polypeptide chain and in either direction (e.g., VL-VH or VH-VL). Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a desired structure for antigen binding. In a preferred embodiment, the CAR contemplated herein comprises an antigen-specific binding domain, which is an scFv and can be a mouse, human, or humanized scFv. Single-chain antibodies can be cloned from the V region genes of hybridomas specific for the desired target. In a particular embodiment, the antigen-specific binding domain is a humanized scFv that binds to a human CXCR5 polypeptide. Illustrative examples of variable heavy chains suitable for constructing anti-CXCR5 CARs contemplated herein include, but are not limited to, the amino acid sequence shown in SEQ ID NO:9. Illustrative examples of variable light chains suitable for constructing anti-CXCR5 CARs contemplated herein include, but are not limited to, the amino acid sequence shown in SEQ ID NO:11.

[0192] Antibodies and Antibody Fragments:

[0193] CAR comprises an extracellular antigen binding domain, and the extracellular antigen binding domain comprises an antibody or antibody fragment that binds to a CXCR5 polypeptide.Therefore, the antibody or antibody fragment of the present invention includes but is not limited to polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single-chain fragments (scFv), single variable fragments (ssFv), single domain antibodies (e.g., VHH fragments from nano antibodies), Fab fragments, F(ab')2 fragments, fragments produced by Fab expression libraries, anti-idiotypic antibodies and epitope binding fragments or any combination thereof, as long as they retain the similar binding properties of the CAR described herein preferably comprising the corresponding CDR or VH and VL regions as described herein.Mini antibodies and multivalent antibodies (e.g., double antibodies, three antibodies, four-valent antibodies and peptide bodies) can also be used in the method of the present invention.The immunoglobulin molecules of the present invention can be any class (i.e., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecules.Therefore, the term antibody as used herein also includes antibodies and antibody fragments contained in the CAR of the present invention produced by modifying complete antibodies or synthesized de novo using recombinant DNA methods.

[0194] As used herein, "antibody" generally refers to a protein consisting of one or more polypeptides that are substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. When the term "antibody" is used, it may also be considered to refer to the term "antibody fragment". Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes, as well as countless immunoglobulin variable region genes. Light chains are divided into κ or λ. Heavy chains are divided into γ, μ, α, δ or ε, which define the classes of immunoglobulins IgG, IgM, IgA, IgD and IgE, respectively. It is known that the basic immunoglobulin (antibody) structural unit comprises a tetramer or a dimer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" (L) chain (about 25 kD) and a "heavy" (H) chain (about 50 to 70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" refer to these variable regions of the light chain and heavy chain, respectively. Optionally, the antibody or immunological portion of the antibody can be chemically conjugated to other proteins or expressed as a fusion protein with other proteins.

[0195] The CAR of the present invention is intended to bind to a mammalian, particularly human, protein target. The use of protein names may correspond to the mouse or human form of the protein.

[0196] The affinity of the binding domain polypeptides and the CAR proteins according to the present disclosure can be readily determined using conventional techniques, such as by competitive ELISA (enzyme-linked immunosorbent assay) or by binding association, or a displacement assay using a labeled ligand, or using a surface plasmon resonance device (e.g., Biacore).

[0197] Any method known in the art can be used to prepare a humanized antibody comprising one or more CDRs of an antibody of the invention or derived from one or more CDRs of the antibody. For example, four general steps can be used to humanize a monoclonal antibody. They are: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light chain and heavy chain variable domains, (2) designing a humanized antibody, i.e., deciding which antibody framework region to use in the humanization process, (3) actual humanization methods / techniques, and (4) transfection and expression of the humanized antibody. See, e.g., U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; 6,180,370; 5,225,539; 6,548,640.

[0198] The term humanized antibody refers to at least a portion of the framework region of an immunoglobulin, and optionally a portion of the CDR region or other regions involved in binding are derived or adjusted to a human immunoglobulin sequence. Humanized, chimeric or partially humanized mouse monoclonal antibodies can be prepared, for example, by recombinant DNA technology from mouse and / or human genomic DNA sequences encoding H and L chains or by cDNA clones encoding H and L chains. Humanized mouse antibodies can be produced by connecting the CDR region of a non-human antibody to a human constant region by recombinant DNA technology (Queen et al., 1989; WO 90 / 07861). Alternatively, the monoclonal antibody used in the method of the present invention can be a human monoclonal antibody. Human antibodies can be obtained, for example, using phage display methods (WO91 / 17271; WO 92 / 01047).

[0199] As used herein, humanized antibodies also refer to non-human (e.g., murine, camel, llama, shark) forms of antibodies that are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.

[0200] As used herein, people or humanized antibodies or antibody fragments refer to antibodies with an amino acid sequence corresponding to an amino acid sequence of an antibody produced by a person and / or an amino acid sequence prepared using any technology for preparing human antibodies known in the art or disclosed herein. Human antibodies or their fragments can be selected by competitive binding experiments, or otherwise selected to have the same epitope specificity as a specific mouse antibody. Surprisingly, the humanized antibodies of the present invention share the useful functional properties of mouse antibodies to a great extent. Human polyclonal antibodies can also be provided in the form of human serum immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using amyloid fibrils and / or non-fibril polypeptides or their fragments as affinity reagents. Monoclonal antibodies can be obtained from serum according to the technology described in WO 99 / 60846.

[0201] Variable regions and CDRs

[0202] The variable region of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable region of both the heavy and light chains consists of four framework regions (FRs) connected by three complementary determining regions (CDRs) (also referred to as hypervariable regions). The CDRs in each chain are held together tightly by the FRs and, together with the CDRs in the other chain, help to form the antigen binding site of the antibody.

[0203] There are many techniques that can be used to determine CDRs, such as methods based on cross-species sequence variability (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda, Maryland); and methods based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al. (1997) J. Molec. Biol. 273:927-948). Alternative methods include the IMGT International ImMunoGeneTics Information System (Marie-Paule Lefranc). The Kabat definition is based on sequence variability and is the most commonly used method. The Chothia definition is based on the position of the structural loop regions, with the AbM definition being a compromise between the two used by the AbM antibody modeling software of Oxford Molecular (see www.bioinf.org.uk: Dr. Andrew CRMartin's group). As used herein, CDRs may refer to CDRs defined by one or more methods or by a combination of these methods.

[0204] In some embodiments, the present invention provides an antibody or fragment thereof incorporated into CAR, wherein the antibody or fragment thereof comprises at least one CDR, at least two, at least three or more CDRs substantially identical to at least one CDR, at least two, at least three or more CDRs of the antibody of the present invention.Other embodiments include antibodies having at least two, three, four, five or six CDRs, which are substantially identical to the antibodies of the present invention or derived from at least two, three, four, five or six CDRs of the antibodies of the present invention.In some embodiments, at least one, two, three, four, five or six CDRs are at least about 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98% or 99% identical to at least one, two, three CDRs of the antibodies of the present invention.It should be understood that for the purposes of the present invention, although the degree of activity may vary (more or less) compared to the antibody, the binding specificity and / or overall activity is generally retained.

[0205] Other Components of CAR

[0206] In certain embodiments, the CAR contemplated herein may include a linker residue between each domain, which is added for the appropriate spacing and configuration of the molecule, such as a linker comprising the following amino acid sequence, which connects the VH and VL domains, and provides a spacer function compatible with the interaction of the two sub-binding domains, so that the resulting polypeptide maintains specific binding affinity to the same target molecule as the antibody comprising the same light chain and heavy chain variable region. The CAR contemplated herein may include one, two, three, four or five or more linkers. In a particular embodiment, the length of the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids or about 10 to about 20 amino acids, or any intermediate length of amino acids.

[0207] Illustrative examples of linkers include glycine polymers; glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art, such as Whitlow linkers. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to act as neutral tethers between domains of fusion proteins (such as CAR described herein).

[0208] In a particular embodiment, the binding domain of the CAR is followed by one or more "spacers" or "spacer polypeptides", which refer to regions that move the antigen binding domain away from the surface of the effector cell to achieve proper cell / cell contact, antigen binding, and activation. In certain embodiments, the spacer region is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer region may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer region comprises the CH2 and CH3 domains of IgG1 or IgG4. In one embodiment, the Fc binding domain of this spacer / hinge region is mutated in a manner that prevents CAR from binding to Fc receptors expressed on macrophages and other innate immune cells.

[0209] In some embodiments, the binding domain of CAR may be followed by one or more "hinge domains", which play a role in positioning the antigen binding domain away from the effector cell surface to achieve appropriate cell / cell contact, antigen binding and activation. CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for CAR described herein include hinge regions derived from the extracellular region of type 1 membrane proteins (e.g., CD8α, CD4, CD28, PD1, CD152 and CD7), and the hinge region may be a wild-type hinge region from these molecules or may be changed. In another embodiment, the hinge domain includes PD1, CD152 or CD8α hinge regions.

[0210] "Transmembrane domain" is a part of CAR that fuses the extracellular binding part and the intracellular signaling domain and anchors CAR to the plasma membrane of immune effector cells. The TM domain can be derived from natural, synthetic, semi-synthetic or recombinant sources. The TM domain can be derived from T cell receptors, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD1 α, β or ζ chain. In one embodiment, the CAR contemplated herein comprises a TM domain derived from CD8α or CD28.

[0211] In a particular embodiment, the CAR considered herein comprises an intracellular signaling domain. "Intracellular signaling domain" refers to a part of CAR that is involved in transducing information of effective anti-CXCR5 CAR bound to human CXCR5 polypeptides to the interior of immune effector cells to trigger effector cell functions, such as activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells or antigen binding to other cell responses caused by the extracellular CAR domain. The term "effector function" refers to the specialized function of immune effector cells. The effector function of T cells can be, for example, cytolytic activity or activity that helps or includes cytokine secretion. Therefore, the term "intracellular signaling domain" refers to a part of the transduction effector function signal of a protein and instructs cells to perform specialized functions. The CAR considered herein comprises one or more costimulatory signaling domains to enhance the efficacy, amplification and / or memory formation of T cells expressing CAR receptors. As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors, and costimulatory molecules provide a second signal required for the effective activation and function of T lymphocytes after binding to antigens.

[0212] In one embodiment, CAR includes an intracellular domain, which includes a co-stimulatory domain and a signal transduction (activation) domain. Therefore, the CAR construct may include an intracellular signal transduction domain (CD3ζ) of a native T cell receptor complex and provide a second signal to stimulate one or more co-stimulatory domains for complete T cell activation. The co-stimulatory domain is considered to increase the production of CAR T cell cytokines and promote T cell replication and T cell persistence. The co-stimulatory domain has also been shown to potentially prevent CAR T cell consumption, increase T cell anti-tumor activity and enhance the survival of CAR T cells in patients. As a non-limiting example, in preclinical studies, the CAR construct with a 4-1BB co-stimulatory domain is associated with progressive continuous expansion and effector function, persistence enhancement, and central memory cells (TCM) enrichment in T cell subsets. 4-1BB is a member of the tumor necrosis factor (TNF) superfamily, and is an inducible glycoprotein receptor expressed in vivo mainly on antigen-activated CD4 and CD8 T cells. As a non-limiting example, CD28 is a member of the immunoglobulin (Ig) superfamily. It is constitutively expressed on resting and activated CD4 and CD8 T cells and plays a key role in T cell activation by stimulating the PI3K-AKT signal transduction pathway. In one embodiment, the intracellular domain contains both 4-1BB and CD28 costimulatory domains. Other costimulatory domains include ICOS and OX40 that can bind to the CD3ζ signaling (activation) domain.

[0213] Peptides

[0214] Unless otherwise indicated, "peptide", "polypeptide", "polypeptide fragment" and "protein" are used interchangeably and in the conventional sense, i.e., as an amino acid sequence. Polypeptides are not limited to a particular length, for example, they may comprise a full-length protein sequence or a fragment of a full-length protein, and may include post-translational modifications of the polypeptide (e.g., glycosylation, acetylation, phosphorylation, etc.), as well as other known modifications that occur naturally and non-naturally.

[0215] In various embodiments, the CAR polypeptide contemplated herein comprises a signal (or leader) sequence at the N-terminus of the protein, which guides the transfer of the protein in a co-translational or post-translational manner. Any of a variety of well-known recombinant and / or synthetic techniques can be used to prepare polypeptides. The polypeptide contemplated herein specifically encompasses CAR of the present disclosure, or a sequence of one or more amino acids of a CAR as disclosed herein that is missing, added and / or substituted.

[0216] As used herein, "isolated peptides" or "isolated polypeptides" etc. refer to peptide or polypeptide molecules that are separated and / or purified in vitro from the cell environment and the association with other components of the cell, i.e., the peptide or polypeptide molecules are not significantly associated with in vivo substances. Similarly, "isolated cells" refer to cells that have been obtained from in vivo tissues or organs and are substantially free of extracellular matrix.

[0217] Nucleic Acids

[0218] As used herein, the term "polynucleotide" or "nucleic acid molecule" refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive strand RNA (RNA (+)), negative strand RNA (RNA (-)), genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotides of the present invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any reference sequence described herein, typically wherein the variant maintains at least one biological activity of the reference sequence. In various illustrative embodiments, the present invention contemplates, in part, polynucleotides including expression vectors, viral vectors and transfer plasmids, as well as compositions and cells comprising them.

[0219] Any of a variety of mature techniques known and available in the art can be used to prepare, manipulate and / or express polynucleotides. In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, autonomous replication sequences and transposable elements. Other exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)), bacteriophages (e.g., lambda phages or M13 phages) and animal viruses. Examples of animal virus categories that can be used as vectors include, but are not limited to, retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses and papovaviruses (e.g., SV40). Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells, pLenti4 / V5-DEST for slow virus-mediated gene transfer and expression in mammalian cells. TM 、pLenti6 / V5-DEST TM and pLenti6.2 / V5-GW / lacZ (Invitrogen). In particular embodiments, the coding sequence of the chimeric protein disclosed herein can be ligated into such an expression vector to express the chimeric protein in mammalian cells. The "control elements" or "regulatory sequences" present in the expression vector are those untranslated regions of the vector - origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence) introns, polyadenylation sequence, 5' and 3' untranslated regions - which interact with host cell proteins for transcription and translation. The strength and specificity of such elements may vary. Depending on the vector system and host used, a number of suitable transcription and translation elements can be used, including ubiquitous promoters and inducible promoters.

[0220] Carrier

[0221] In a particular embodiment, cells (e.g., immune effector cells, such as T cells) are transduced with a retroviral vector (e.g., a lentiviral vector) encoding CAR. For example, immune effector cells are transduced with a vector encoding CAR, the CAR comprising humanized anti-CXCR5 antibodies or antigen binding fragments in combination with CXCR5 polypeptides and transmembrane domains and intracellular signaling domains, so that these transduced cells can induce CAR-mediated cytotoxic responses.

[0222] Retrovirus is a common tool for gene delivery. In a particular embodiment, retrovirus is used to deliver polynucleotides encoding chimeric antigen receptors (CAR) to cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes the genomic RNA of the virus into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus". Provirus acts as a template for RNA polymerase II and guides the expression of RNA molecules that encode structural proteins and enzymes required to produce new viral particles.

[0223] Illustrative retroviruses suitable for use in particular embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), Gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Flinder's murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0224] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Vesnamedi virus (VMV) virus; Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (i.e., HIV cis-acting sequence elements) are preferred. In a particular embodiment, lentivirus is used to deliver a polynucleotide comprising CAR to a cell.

[0225] The term "vector" as used herein refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is usually connected to a carrier nucleic acid molecule, such as an insertion into a carrier nucleic acid molecule. The vector may include a sequence that directs autonomous replication in the cell, or may include a sequence sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses. In other embodiments of the present invention, CrispR / Cas and TALEN-mediated insertion of CXCR5 CAR encoding nucleic acids can be used. Vectors suitable for CrispR / Cas and TALEN-mediated insertion are known to technicians.

[0226] It will be apparent to those skilled in the art that the term "viral vector" is used broadly to refer to nucleic acid molecules (e.g., transfer plasmids) that contain nucleic acid elements of viral origin that typically facilitate transfer or integration of nucleic acid molecules into the cellular genome, or to viral particles that mediate nucleic acid transfer. Viral particles will typically contain various viral components and sometimes host cell components in addition to nucleic acid.

[0227] The term viral vector may refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the nucleic acid itself that is transferred. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements primarily derived from retroviruses, or portions thereof.

[0228] Therefore, in a preferred embodiment, the present invention relates to a method for transfecting cells with an expression vector encoding CAR. For example, in some embodiments, the vector comprises additional sequences, such as sequences that promote CAR expression, such as promoters, enhancers, poly-A signals and / or one or more introns. In a preferred embodiment, the flank of the sequence encoding CAR is a transposon sequence, so that the presence of a transposase allows the coding sequence to be integrated into the genome of the transfected cell.

[0229] In some embodiments, the genetically transformed cells are further transfected with a transposase that promotes the integration of the sequence encoding CAR into the genome of the transfected cells. In some embodiments, the transposase is provided as a DNA expression vector. However, in a preferred embodiment, the transposase is provided in the form of expressible RNA or protein so that the transposase is not expressed for a long time in transgenic cells. For example, in some embodiments, the transposase is provided in the form of mRNA (e.g., mRNA comprising a cap and a poly-A tail). According to an embodiment of the present invention, any transposase system can be used. However, in some embodiments, the transposase is a salmonid Tel-like transposase (SB). For example, the transposase can be a so-called "Sleeping Beauty" transposase, see, for example, U.S. Patent No. 6,489,458, which is incorporated herein by reference. In some embodiments, the transposase is an engineered enzyme with increased enzymatic activity. Some specific examples of transposases include, but are not limited to, SB 10, SB 11, or SB 100X transposases (see, e.g., Mates et al., 2009, Nat Genet. 41(6):753-61 or US9228180, which are incorporated herein by reference. For example, the method may comprise electroporating cells with mRNA encoding SB 10, SB 11, or SB 100X transposase.

[0230] Sequence variants:

[0231] Also included within the scope of the present invention are sequence variants (e.g., sequence variants defined by % sequence identity) of nucleic acids, proteins, antibodies, antibody fragments, and / or CARs that maintain similar binding properties of the present invention. Such variants display alternative sequences, but substantially maintain the same binding properties (e.g., target specificity), as provided specific sequences are referred to as functional analogs or functionally similar. Sequence identity relates to the percentage of identical nucleotides or amino acids when performing sequence alignment.

[0232] As used herein, "sequence identity" refers to the degree to which sequences are identical on a nucleotide-to-nucleotide basis or an amino acid-to-amino acid basis in a comparison window. Therefore, "sequence identity percentage" can be determined by comparing two optimally aligned sequences in a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) exists simultaneously in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and then multiplying the result by 100 to obtain the sequence identity percentage. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, typically wherein the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0233] Those of ordinary skill in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides have minimal homology or sequence identity to the nucleotide sequence of any natural gene. However, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Also included in the present invention are deletions, substitutions and other changes in sequences that are classified as being identical to the sequence.

[0234] Also included within the scope of the present invention are modifications of protein sequences that can occur by substitution. Substitution, as defined herein, is a modification of the amino acid sequence of a protein in which one or more amino acids are replaced by the same number of (different) amino acids, thereby producing a protein comprising an amino acid sequence different from that of the primary protein. Substitutions that preferably do not significantly change the function of the protein can be performed. Like additions, substitutions can be natural or artificial. It is well known in the art that amino acid substitutions can be performed without significantly changing the function of a protein. This is especially true when the modification involves a "conservative" amino acid substitution, in which one amino acid is replaced by another amino acid with similar properties. Such "conservative" amino acids can be natural or synthetic amino acids that can be replaced without significantly affecting the structure and function of the protein due to size, charge, polarity and conformation. Typically, many amino acids can be replaced by conservative amino acids without adversely affecting the function of the protein.

[0235] In general, the nonpolar amino acids Gly, Ala, Val, Ile and Leu; the nonpolar aromatic amino acids Phe, Trp and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gln, Asn and Met; the positively charged amino acids Lys, Arg and His; the negatively charged amino acids Asp and Glu represent several groups of conserved amino acids. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can be substituted for each other even though they belong to different groups.

[0236] Substitution variants have removed at least one amino acid residue in the antibody molecule and inserted a different residue at that position. The most interesting sites for substitution mutagenesis include hypervariable regions, but FR changes are also considered. If such substitutions result in changes in biological activity, then more substantial changes referred to as "exemplary substitutions" in the following table or as further described below with reference to amino acid classes may be introduced and the product screened.

[0237] Potential amino acid substitutions:

[0238]

[0239]

[0240] Substantial modification of the biological properties of the antibody is achieved by selecting substitutions that have a significant effect on maintaining: (a) the structure of the polypeptide backbone in the area of ​​the substitution, such as a sheet or helical configuration, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the number of side chains.

[0241] Conservative amino acid substitutions are not limited to naturally occurring amino acids, but also include synthetic amino acids. Commonly used synthetic amino acids are: omega amino acids of various chain lengths and cyclohexylalanine, which are neutral non-polar analogs; citrulline and methionine sulfoxide, which are neutral non-polar analogs; phenylglycine, which is an aromatic neutral analog; cysteic acid, which is a negatively charged analog; ornithine, which is a positively charged amino acid analog. As with the naturally occurring amino acids, this list is not exhaustive, but merely examples of substitutions well known in the art.

[0242] Genetically modified cells and immune cells

[0243] In a particular embodiment, the present invention contemplates cells genetically modified to express the CAR considered herein, for the treatment of B cell-related disorders. As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in the cell. The terms "genetically modified cells", "modified cells" and "redirected cells" are used interchangeably. As used herein, the term "gene therapy" refers to the permanent or temporary introduction of additional genetic material in the form of DNA or RNA into the total genetic material in the cell, to restore, correct or modify the expression of genes, or for the purpose of expressing therapeutic polypeptides (such as CAR). In a particular embodiment, the CAR considered herein is introduced into immune effector cells and expressed in immune effector cells, so as to redirect the specificity of immune effector cells to the target antigen of interest, such as CXCR5 polypeptides.

[0244] "Immune cell" or "immune effector cell" is any immune system cell with one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). Immune effector cells can also be distinguished from iPSCs (induced pluripotent stem cells).

[0245] The immune effector cells of the present invention can be autologous / autogeneic ("self") or non-autologous ("non-self", such as allogeneic, isogenic or xenogeneic). As used herein, "autologous" refers to cells from the same subject and represents a preferred embodiment of the present invention. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the cells being compared.

[0246] As used herein, "isogenic" refers to cells from a different subject that are genetically identical to the cells being compared. As used herein, "xenogeneic" refers to cells of a different species than the cells being compared. In preferred embodiments, the cells of the invention are autologous or allogeneic.

[0247] Illustrative immune effector cells used with the CAR considered herein include T lymphocytes. The term "T cell" or "T lymphocyte" is recognized in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells) or activated T lymphocytes. Cytokine-induced killer (CIK) cells are generally natural killer (NK)-like T lymphocytes restricted by non-major histocompatibility complex (MHC) positive for CD3 and CD56. T cells can be helper T cells (Th; CD4+T cells), such as helper T cells 1 (Th1) or helper T cells 2 (Th2). T cells can be cytotoxic T cells (CTL; ​​CD8+T cells), CD4+CD8+T cells, CD4CD8 T cells or any other T cell subsets. Other exemplary populations of T cells suitable for particular embodiments include naive T cells and memory T cells and stem cell-like memory cells (TSCM).

[0248] For example, when reintroduced into a patient after autologous cell transplantation, T cells modified with the CAR of the present invention as described herein can recognize and kill tumor cells. Compared with other T cells, CIK cells can have enhanced cytotoxic activity and therefore represent a preferred embodiment of the immune cells of the present invention.

[0249] As will be understood by those skilled in the art, other cells may also be used as immune effector cells together with CAR as described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include progenitor cells of effector cells, wherein such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Progenitor cells can be iPSCs that become immune effector cells under limited culture conditions.

[0250] The present invention provides a method for preparing immune effector cells expressing CAR considered herein. In one embodiment, the method includes transfection or transduction of immune effector cells separated from an individual so that the immune effector cells express one or more CARs as described herein. In certain embodiments, immune effector cells are separated from an individual and genetically modified without further in vitro operation. Such cells can then be directly re-administered to an individual. In a further embodiment, before being genetically modified to express CAR, immune effector cells are first activated and stimulated to proliferate in vitro. In this regard, immune effector cells can be cultured before and / or after genetic modification (i.e., transduced or transfected to express CAR considered herein).

[0251] In a particular embodiment, before the in vitro manipulation or genetic modification of the immune effector cells described herein, the cell source is obtained from the subject. In a particular embodiment, the CAR-modified immune effector cells include T cells. T cells can be obtained from a variety of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using a variety of techniques known to those skilled in the art, such as precipitation (e.g., FICOLL TM separation), antibody-conjugated bead-based methods (e.g. MACS TM Separation (Miltenyi)). In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product generally contains lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma portion, and the cells are placed in an appropriate buffer or culture medium for subsequent processing. The cells can be washed with PBS or other suitable solutions lacking calcium, magnesium and most (if not all other) divalent cations. As will be appreciated by those of ordinary skill in the art, the washing steps can be accomplished by methods known to those skilled in the art, such as by using a semi-automatic flow-through centrifuge. For example, Cobe 2991 unit processor, Baxter CytoMate, etc. After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions containing or not containing buffers. In certain embodiments, the undesirable components of the apheresis sample can be removed in the directly resuspended cell culture medium.

[0252] In certain embodiments, the erythrocytes are lysed and the monocytes are depleted, for example by PERCOLL TM Gradient centrifugation is used to separate T cells from peripheral blood mononuclear cells (PBMC). The specific subpopulation of T cells can be further separated by positive or negative selection techniques. One method used herein is to sort and / or select cells by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on cells selected negatively.

[0253] PBMC can be directly genetically modified to express CAR using the methods considered herein. In some embodiments, after separating PBMC, T lymphocytes are further separated, and in some embodiments, cytotoxic and helper T lymphocytes can be sorted into immature, memory and effector T cell subsets before or after genetic modification and / or amplification. CD8+ cells can be obtained by using standard methods. In some embodiments, CD8+ cells are further classified into immature, central memory and effector cells by identifying cell surface antigens associated with each of those types of CD8+ cells.

[0254] In some embodiments, the immune cells of the invention (eg, T cells described herein) can be obtained from induced pluripotent stem cells (iPSCs) using methods known to those of skill.

[0255] The acceptance method of producing CAR T cells depends on the genetic modification and amplification of mature circulating T cells. Such a process utilizes autologous T cells, and is expressed by endogenous TCR and rejected by MHC incompatibility, reducing the risk of allogeneic T cells developing graft-versus-host disease (GvHD). As an alternative, direct differentiation of engineered T cells in vitro from pluripotent stem cells (such as induced pluripotent stem cells) provides a substantially unlimited cell source that can be genetically modified to express the CAR of the present invention. In some embodiments, the so-called main iPSC system can be maintained, which represents a renewable source that can consistently and repeatedly produce homogeneous cell products. In some embodiments, before amplification and differentiation into desired immune cells, preferably T cells, it is considered to transform the main iPSC cell line with nucleic acids encoding CAR. T lymphocytes can be produced, for example, from iPSC, so that iPSC can be modified with nucleic acids encoding CAR, then amplified and differentiated into T cells to be applied to patients. Suitable immune cells (such as T cells) can also be differentiated from iPSC, then transformed with nucleic acids encoding CAR, and then amplified before administration. All possible combinations of iPSC expansion, genetic modification, and expansion to provide a suitable number of cells for administration are contemplated in the present invention.

[0256] Immune effector cells (such as T cells) can be genetically modified after being separated using known methods, or immune effector cells can be activated and expanded in vitro (or differentiated in the case of progenitor cells) before being genetically modified. In a particular embodiment, immune effector cells (such as T cells) are genetically modified with chimeric antigen receptors considered herein (such as transduced with a viral vector comprising a nucleic acid encoding CAR), and then activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express CAR using methods described in, for example, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0257] In another embodiment, a donor population of immune effector cells can be genetically modified using, for example, a mixture of one, two, three, four, five or more different expression vectors, wherein each vector encodes a different chimeric antigen receptor protein as contemplated herein. The resulting modified immune effector cells form a mixed population of modified cells, wherein a portion of the modified cells express more than one different CAR protein.

[0258] In one embodiment, the present invention provides a method for storing immune effector cells of genetically modified mice, people or humanized CAR proteins targeting CXCR5 proteins, the method including cryopreservation of immune effector cells so that the cells remain viable after thawing. A portion of immune effector cells expressing CAR proteins can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with B cell-related disorders. When necessary, the cryopreserved converted immune effector cells can be thawed, grown and expanded into more such cells.

[0259] Compositions and preparations

[0260] As considered herein, the compositions considered herein may include one or more polypeptides, polynucleotides, vectors comprising them, genetically modified immune effector cells, etc. Compositions include, but are not limited to, pharmaceutical compositions." pharmaceutical compositions "refers to compositions prepared in pharmaceutically acceptable or physiologically acceptable solutions, which are used to be applied to cells or animals individually or in combination with one or more other treatment modalities. It should also be understood that, if necessary, the compositions of the present invention may also be administered in combination with other agents (e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents). In fact, there is no restriction on other components that may also be included in the composition, provided that other agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0261] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0262] As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; gum tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and any other compatible substances used in pharmaceutical formulations.

[0263] In a particular embodiment, the composition of the present invention includes a certain amount of CAR-expressing immune effector cells considered herein. As used herein, the term "amount" refers to an "effective amount (amount effective / effective amount)" of a genetically modified therapeutic cell (e.g., T cell) to achieve a beneficial or desired prevention or therapeutic result including a clinical result.

[0264] A "prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve the desired prophylactic outcome. Typically, but not necessarily, a prophylactic dose is less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to disease or in the early stages of disease. The term prophylactic does not necessarily refer to a complete prohibition or prevention of a particular medical disease. The term prophylactic also refers to a reduction in the risk of a medical disease developing or worsening its symptoms.

[0265] A "therapeutically effective amount" of genetically modified therapeutic cells can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of stem cells and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the virus or transduced therapeutic cells. The term "therapeutically effective amount" includes an amount that effectively "treats" a subject (e.g., a patient). When indicating a therapeutic amount, a physician can determine the exact dose of the composition of the invention to be administered, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and patient (subject) condition.

[0266] Generally, it can be provided that the pharmaceutical composition comprising the T cells described herein can be taken at 10 2 Up to 10 10 cells / kg body weight, preferably 10 5 Up to 10 7 The amount of cells will depend on the intended end use of the composition, and the type of cells included in the composition will also depend on the end use. For the uses provided herein, the cells are typically in a volume of 1 liter or less, which may be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the desired cell density is typically greater than 10 6 cells / mL, usually greater than 10 7 cells / mL, usually greater than 10 8 Clinically relevant numbers of immune cells can be distributed into multiple infusions that cumulatively equal or exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12In some aspects of the invention, a smaller number of cells may be administered, particularly since all infused cells will be redirected to a specific target antigen. The CAR-expressing cell composition may be administered multiple times at doses within these ranges. The cells may be allogeneic, isogenic, xenogeneic, or autologous to the patient being treated.

[0267] Typically, compositions comprising cells activated and amplified as described herein can be used to treat and prevent diseases occurring in immunocompromised individuals. In particular, compositions comprising CAR-modified T cells contemplated herein are used to treat B cell malignancies. The CAR-modified T cells of the present invention can be administered alone, or as a pharmaceutical composition with a carrier, diluent, excipient and / or in combination with other ingredients (eg, IL-2 or other cytokines) or cell populations. In a particular embodiment, the pharmaceutical composition contemplated herein comprises a certain amount of genetically modified T cells combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0268] The pharmaceutical composition of the present invention comprising an immune effector cell group (such as T cell) expressing CAR, may include a buffer, such as neutral buffered saline, phosphate buffered saline, etc.;Carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol;Protein;Polypeptide or amino acid, such as glycine;Antioxidant;Chelating agent, such as EDTA or glutathione;Adjuvant (such as aluminum hydroxide);And preservative. The composition of the present invention is preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

[0269] Liquid pharmaceutical compositions, whether solutions, suspensions or other similar forms, may include one or more of the following: sterile diluents, such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils (e.g., synthetic polyglycerol monoesters or diglycerides that can be used as solvents or suspension media), polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for adjusting tonicity, such as sodium chloride or glucose. Parenteral preparations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0270] In a specific embodiment, the composition considered herein comprises an effective amount of expression CAR immune effector cells alone or in combination with one or more therapeutic agents.Therefore, the immune effector cell composition expressing CAR can be applied alone or in combination with other known cancer treatments (such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc.).The composition can also be applied in combination with antibiotics.Such therapeutic agents can be accepted in the art as standard treatments for specific disease states (such as specific cancers) described herein.Exemplary therapeutic agents considered include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapy, radiotherapy, therapeutic antibodies or other active agents and adjuvants.

[0271] Treatment

[0272] The genetically modified immune effector cells contemplated herein provide an improved approach to adoptive immunotherapy for the treatment of medical conditions associated with the presence of pathogenic cells expressing CXCR5, including, but not limited to, immunoregulatory disorders and hematological malignancies.

[0273] As used herein, "a medical condition associated with the presence of pathogenic cells expressing CXCR5" refers to a medical condition such as cancer or an autoimmune disease, wherein cells associated with the pathophysiology of the disease show expression of CXCR5, and preferably show CXCR5 presented on the cell surface. The expression of CXCR5 can be determined by a variety of methods known to the skilled person, for example, by isolating cells from a patient and evaluating these cells by PCR using primers for CXCR5 transcripts, immunostaining with anti-CXCR5 antibodies, or analysis by flow cytometry. Such pathogenic cells can typically be pathogenic mature B cells and / or memory B cells and / or pathogenic T cells and / or follicular helper T cells.

[0274] In particular embodiments, the compositions comprising CAR-modified T cells contemplated herein are used to treat hematological malignancies, including but not limited to B-cell malignancies, such as non-Hodgkin lymphoma (NHL) with or without leukemic tumor cell dissemination (e.g., B-cell NHL or T-cell non-Hodgkin lymphoma).

[0275] Non-Hodgkin lymphoma encompasses a large group of cancers of the lymphocytes (white blood cells). Non-Hodgkin lymphoma can occur at any age and is usually characterized by larger-than-normal lymph nodes, fever, and weight loss. Non-Hodgkin lymphoma can also arise in extranodal sites, such as the central nervous system and mucosal tissues including the lungs, intestines, colon, and internal organs. There are many different types of non-Hodgkin lymphoma. For example, non-Hodgkin lymphoma can be divided into aggressive (fast-growing) and indolent (slow-growing) types.

[0276] Non-Hodgkin lymphoma can be derived from B cells and T cells. As used herein, the term "non-Hodgkin lymphoma" includes "B cell" and "T cell" non-Hodgkin lymphoma. B cell non-Hodgkin lymphoma (NHL) includes Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma and mantle cell lymphoma. Lymphomas that occur after bone marrow or stem cell transplantation are usually B cell non-Hodgkin lymphoma.

[0277] T-cell lymphomas account for approximately 15% of all NHLs in the United States. There are many different forms of T-cell lymphoma, such as angioimmunoblastic T-cell lymphoma (AITL), which is a mature T-cell lymphoma of the blood or lymphangiogenic immunoblasts. Other forms of T-cell lymphoma involve cutaneous T-cell lymphoma and T-cell lymphoma with leukemic disseminated.

[0278] Chronic lymphocytic leukemia (CLL), which can also be treated with this CAR, is an indolent (slow-growing) cancer that causes a slow increase in immature white blood cells (B lymphocytes). The cancer cells spread through the blood and bone marrow and can also affect the lymph nodes or other organs, such as the liver and spleen. CLL eventually leads to bone marrow failure. Another form of the disease is called small lymphocytic lymphoma, which is mainly distributed in secondary lymphoid organs, such as the lymph nodes and spleen.

[0279] In one embodiment of the present invention, CAR or an immune cell expressing said CAR is intended for use in the treatment of an autoimmune disease, preferably an autoantibody-dependent autoimmune disease, preferably an autoimmune disease with an inflammatory component.

[0280] Deregulated CXCR5-expressing follicular helper T cells (Tfh) represent an important mechanism that contributes to increased humoral immune responses and autoantibody production in autoimmune diseases. Therefore, in the context of autoimmunity, CXCR5-expressing Tfh cells represent a viable target.

[0281] The autoimmune disease to be treated is preferably selected from Takayasu's arteritis, giant cell arteritis, familial Mediterranean fever, Kawasaki disease, polyarteritis nodosa, cutaneous polyarteritis nodosa, hepatitis-associated arteritis, Behcet's syndrome, Wegener's granulomatosis, ANCA vasculitis, Churg-Strauss syndrome,Microscopic polyangiitis, connective tissue vasculitis, Hennoch-purpura, cryoglobulin vasculitis, cutaneous leukocytoclastic vasculitis, tropical aortitis, sarcoidosis, Cogan's syndrome, Wiskott-Aldrich syndrome, leprosarcoma nodosa arteritis, primary CNS vasculitis, occlusive thrombotic vasculitis, paraneoplastic arteritis, urticaria, Dego's disease, myelodysplastic syndrome, erythema protuberans, hyperimmunoglobulin D, allergic rhinitis, bronchial asthma, chronic obstructive pulmonary disease, periodontitis, rheumatoid arthritis, atherosclerosis, amyloidosis, Morbus Chron, ulcerative colitis, autoimmune myositis, diabetes mellitus, Guillain-Barré syndrome Syndrome), histiocytosis, osteoarthritis, atopic dermatitis, periodontitis, chronic sinusitis, psoriasis, psoriatic arthritis, microscopic colitis, pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, erythema nodosum, otitis, cryoglobulinemia, Sjogren's syndrome syndrome), lupus erythematosus (preferably systemic lupus erythematosus (SLE)), aplastic anemia, myelofibrosis, chronic inflammatory demyelinating polyneuropathy, Kimura's disease, systemic sclerosis, chronic periarthritis, chronic prostatitis, idiopathic pulmonary fibrosis, chronic granulomatous disease, idiopathic achalasia, bleomycin-induced lung inflammation, cytarabine-induced lung inflammation, autoimmune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune lymphopenia, Chagas' disease, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's thyroiditis, atrophic thyroiditis, Graves' disease, autoimmune polyglandular syndrome, autoimmune Addison's syndrome, pemphigus vulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmune alopecia, vitiligo, antiphospholipid syndrome, myasthenia gravis, stiff-man syndrome syndrome, Goodpasture's syndrome, sympathetic ophthalmia, folliculitis, Sharp syndrome and / or Evans syndrome, in particular hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and most preferably SLE.

[0282] Systemic lupus erythematosus (SLE), also known as lupus, is an autoimmune disease in which the body's immune system attacks healthy tissue in various parts of the body. Symptoms vary from person to person and can range from mild to severe. Common symptoms include joint pain and swelling, fever, chest pain, hair loss, mouth sores, swollen lymph nodes, feeling tired, and a red rash, most commonly on the face.

[0283] Follicular helper T cells (Tfh) cells have recently been found to be the main cell reservoir of human immunodeficiency virus (HIV) in CD4+ helper T cells (Leong et al., 2017, Frontiers in Immunology, 8: 622), and the CAR immune cells expressing the CAR of the present invention can target the follicular reservoir of helper T cells that produce HIV. In this way, the CAR of the present invention can be used to eliminate or inhibit the reservoir of helper T cells that produce HIV in the treatment of HIV. Therefore, in one embodiment, the reservoir of helper T cells that produce HIV is considered to be a group of pathogenic cells expressing CXCR5, particularly pathogenic T cells.

[0284] As used herein, the terms "individual" and "subject" are generally used interchangeably and refer to any animal showing symptoms of a disease, disorder or condition that can be treated with the gene therapy vectors, cell-based therapeutic agents and methods disclosed elsewhere herein. In a preferred embodiment, the subject includes any animal showing symptoms of a disease, disorder or condition (e.g., B cell malignancy) of the hematopoietic system that can be treated with the cell-based therapeutic agents and methods disclosed herein. Suitable subjects include experimental animals (e.g., mice, rats, rabbits or guinea pigs), farm animals and livestock or pets (e.g., cats or dogs). Non-human primates are included, and preferably human patients are included. Typical subjects include human patients with B cell malignancies, who have been diagnosed with B cell malignancies or who are at risk of having B cell malignancies.

[0285] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even the smallest reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally involve alleviation or amelioration of the symptoms of a disease or condition, or delay in the progression of the disease or condition. "Treatment" does not necessarily mean complete eradication or cure of a disease or condition or its associated symptoms.

[0286] As used herein, "prevent" and similar words, such as "prevented / preventing" or "prophylactic", etc., refer to methods of preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevent" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0287] In one embodiment, a method for treating a B cell-related disorder in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising genetically modified immune effector cells contemplated herein. The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

[0288] The administration of the compositions contemplated herein can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, the compositions are administered parenterally. The phrase "parenteral administration" as used herein refers to the mode of administration except enteral and topical administration, usually by injection, including but not limited to intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node or site of infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0289] The present invention is illustrated by the following figures. These figures should be considered as further descriptions of possible and potentially preferred embodiments, which enhance support of one or more non-limiting embodiments of the present invention.

[0290] Brief description of the drawings:

[0291] Figure 1 : Schematic diagram of the preferred CAR structure.

[0292] Figure 2 : Schematic diagram of preferred CAR constructs H28, R28, HBB1, HBB2 and H28BB.

[0293] Figure 3 : List of preferred constructs and potential combinations of various structural elements of CAR as described herein.

[0294] Figure 4 : Sequence comparison between mAb binding region and the preferred humanized sequence employed in this CAR.

[0295] Figure 5 : Sequence depiction of the DNA sequence encoding the mAb binding region hHC.

[0296] Figure 6 : Sequence depiction of the DNA sequence encoding the mAb binding region hLC.

[0297] Figure 7 : GeneArtTM plasmids with humanized and rat CXCR5-CAR sequences.

[0298] Figure 8 : Gel electrophoresis of constructs and vectors after restriction digestion.

[0299] Fig. 9 : Flow cytometric confirmation of CXCR5 CAR expression on human T cells after retroviral transduction.

[0300] Fig.10 : CXCR5 expression on cell types assessed in functional assays.

[0301] Fig.11 : Co-culture of CAR-transduced human T cells with different target cell lines showed activation of specific T cells by different CXCR5+ B-NHL and control cell lines.

[0302] Fig.12 : Cytotoxicity assays revealed selective killing of CXCR5-positive cell lines.

[0303] Fig.13 : CXCR5-redirected CAR-T cells are effective against B-cell non-Hodgkin lymphoma (B-NHL) in a xenograft NSG mouse model.

[0304] Fig.14: Co-culture of CAR-transduced human T cells with different target cell lines showed activation of specific T cells by different CXCR5+ B-NHL and control cell lines.

[0305] Fig.15 : Co-culture of CAR-transduced human T cells with CXCR5-negative primary cells from different human tissues as targets showed no off-target T-cell activation, whereas the CXCR5-expressing B-NHL cell line JeKo-1 mediated specific T-cell activation and was used as a positive control.

[0306] Detailed description of the attached drawings:

[0307] Figure 1 : Schematic diagram of a preferred CAR structure. The VL and VH domains of the antigen binding domain are depicted, including a linker between the VH and VL domains. A spacer between the antigen binding domain and the transmembrane domain is also depicted. Variants of the intracellular domain are also depicted, including, for example, costimulatory and activation domains.

[0308] Figure 2 : Schematic diagram of preferred CAR constructs H28, R28, HBB1, HBB2 and H28BB. Preferred constructs of the present invention include variants of transmembrane domains, costimulatory domains and activation domains. In essence, preferred embodiments of the present invention can exchange these various domains, preferably those particular embodiments disclosed herein, although other domains with similar functions known to those skilled in the art are also encompassed.

[0309] Figure 3 : List of preferred constructs and potential combinations of various structural elements of CAR as described herein.

[0310] Figure 4 : Sequence comparison between mAb binding region and preferred humanized sequences used in this CAR. An alignment is depicted showing sequence identity between rat and humanized hHC and hLC sequences. As depicted, there is 89% sequence identity between humanized and rat hHC sequences and 93% sequence identity between humanized and rat hLC sequences.

[0311] Figure 5 : Sequence depiction of the DNA sequence encoding the mAb binding region hHC, specifically showing the sequence comparison between the original and codon-optimized (CO) DNA sequences encoding the humanized hHC.

[0312] Figure 6 : Sequence depiction of the DNA sequence encoding the mAb binding region hLC, specifically showing the sequence comparison between the original and codon-optimized (CO) DNA sequences encoding the humanized hLC.

[0313] Figure 7 : GeneArt with humanized and rat CXCR5-CAR sequences TM Plasmid. Cleavage of the scFV was demonstrated using gel electrophoresis.

[0314] Figure 8 : Gel electrophoresis of constructs and vectors after restriction digestion. Plasmid MP71 containing the construct encoding CAR is also depicted.

[0315] Fig. 9 : Flow cytometric confirmation of CXCR5 CAR expression on human T cells after retroviral transduction: CAR expression, construct H28, SP6, not transduced.

[0316] Fig.10 :(A) CXCR5 expression on certain cell types evaluated in functional assays. The B-NHL cell lines DOHH-2, SU-DHL4, OCI-Ly7 and JeKo-1, the main patient-derived MCL xenografts, the B-ALL cell lines NALM6, REH, the MM cell line NCI-H929 and the T-ALL cell line Jurkat were evaluated. From the analysis, it can be seen that CXCR5 is expressed in B-NHLDOHH-2, SU-DHL4, OCI-Ly7 and JeKo-1 cell lines. (B) Excluding the CXCR5 CAR-T cells of the present invention showed cross-reactivity with human healthy tissues, the expression of CXCR5 was evaluated in a group of primary cells derived from human healthy tissues. None of the primary human cells tested (HUVEC, human umbilical vein endothelial cells; HUAEC, human umbilical artery endothelial cells; HA, human astrocytes; HN, human neurons; HPNC, human pericytes; HCoEpiC, human colon epithelial cells) showed CXCR5 surface expression by anti-CXCR5 immunostaining and flow cytometric analysis. (C) CXCR5 density per cell was quantified using QuantiBRITE PE calibration beads and a CXCR5-specific antibody on selected B-NHL cell lines (SU-DHL4, OCI-Ly7, DOHH-2, SC-1, JeKo-1, MEC-1, JVM-3), on a MM cell line (NCI-H929), on B-ALL (REH, NALM-6) and T-ALL (Jurkat) cell lines, on a colon adenocarcinoma cell line (SW-620), and on embryonic kidney cell lines that were either untransfected or transfected with CXCR5 (HEK293, HEK-CXCR5, respectively).

[0317] Fig.11: Co-cultures of human T cells transduced by CAR and different target cell lines show activation of specific T cells by different CXCR5+B-NHL and control cell lines. Functional in vitro co-culture and IFN-γ ELISA were performed. The level of released IFN-γ indicates T cell activation. Untransduced T cells (UT; left column of each series), T cells expressing CXCR5-CAR (CXCR5 (H28); middle column of each series) and SP6 T cells (SP6; right column of each series) were co-cultured with target cell lines DOHH-2, SU-DHL4, OCI-Ly7, JeKo-1, NALM6, REH, NCI-H929 and Jurkat cells. DOHH-2, SU-DHL4, OCI-Ly7 and JeKo-1 target cells show specific IFN-γ release in response to CXCR5 CAR-T cell therapy of the present invention. JVM-3 also shows IFN-γ release after treatment.

[0318] Fig.12 :Cytotoxicity assays revealed selective killing of CXCR5-positive cell lines; essentially no killing was observed in CXCR5-negative cell lines. Two independent functional in vitro co-culture and 51Cr release assays were performed. (A, B) DOHH-2, SU-DHL4, OCI-Ly7, SC-1 (only in (B)), JeKo-1 target cell lines showed lysis after treatment with the CAR-T of the present invention, while cells that do not express CXCR5 (Nalm6 and NCI-H929) did not show lysis.

[0319] Fig.13 : In a xenograft NSG mouse model, CXCR5 redirected CAR-T cells were effective against B-cell non-Hodgkin lymphoma (B-NHL). To provide proof of concept, the potent in vitro activity of CXCR5 CAR-modified T cells was translated into potent antitumor activity in vivo. scid Iq tm1 Wjl / SzJ(NSG) mice cohort was treated with 5×10 5 Mantle cell lymphoma (MCL) cells JeKo-1 were inoculated intravenously ( Fig.13A to C in the figure) and transduced with the luciferase gene together with GFP. NSG mice do not develop T, B, and NK cells and are therefore suitable for tolerance and growth of xenografted human cells. (A) MCL tumors were implanted in the xenograft NSG mouse model. Mice were challenged by intravenous transplantation of MCL cells. On day 5 after tumor inoculation, tumor cell growth was observed by IVIS imaging. To measure the onset of tumor burden, imaging was extended to 120 seconds (day 0). (B) To track the effect of treatment and reduce the bioluminescence intensity for better representation, the mice in (A) were imaged again for 10 seconds on day 0. On the same day, mice were treated with 3×10 6 Mice (n=4) transduced with anti-CXCR5 CAR-transduced T cells and mice (n=3) transduced with SP6-CAR as negative control (day 0). Subsequent IVIS exposure after completion of CAR-T cell transfer at 10 seconds for better comparison between day 0 and day 19. (C) The average values ​​of bioluminescent signals obtained from the target area covering the whole body of each mouse were plotted for each group at each time point. Fig.13 C). Essentially all animals treated with SP6 CAR developed progressive lymphomatous disease characterized by strong luminescent signals in the bone marrow of the hind limbs, thorax, and abdominal organs, whereas this was clearly not the case in the CXCR5 CAR-treated group. This provides the first preclinical in vivo demonstration that CXCR5 CAR-T cells have antitumor activity against B-NHL lymphoma entities.

[0320] Fig.14 : CAR transduced human T cells and co-cultures of different target cell lines show activation of specific T cells by different CXCR5+B-NHL and control cell lines. Functional in vitro co-culture and IFN-γ (upper figure), IL-2 (middle figure) and TNF-α (lower figure) ELISA were performed. The levels of released IFN-γ, IL-2 and TNF-α indicate T cell activation and T cell function. Untransduced (UT, hollow column), T cells expressing CXCR5-CD28 CAR (H28, red column), CXCR5-41BB (HBB1; blue column), CXCR5-CD28 / 41BB (H28BB, green column) and SP6 T cells (SP6, gray column) were co-cultured with target cell lines JeKo-1, DOHH-2, SU-DHL4, OCI-Ly7, REH and NCI-H929 cells. DOHH-2, SU-DHL4, OCI-Ly7 and JeKo-1 target cells showed specific IFN-γ, IL-2 and TNF-α release in response to treatment with the CXCR5 CAR-T cells of the present invention.

[0321] Fig.15: Co-culture of CXCR5-negative primary cells of different human tissues as targets by CAR transduction of human T cells does not show off-target T cell activation, while the B-NHL cell line JeKo-1 expressing CXCR5 mediates specific T cell activation and is used as a positive control. Functional in vitro co-culture and IFN-γ ELISA were performed. Untransduced (UT, hollow column), T cells expressing CXCR5-CD28 CAR (H28, red column) and SP6 CAR T cells (SP6, gray column) were co-cultured with primary cells HUVEC, HUAEC, HA, HN, HPNC, HCoEpiCs, and T-ALL cell line Jurkat, and B-NHL cell line JeKo-1. Lack of IFN-γ release indicates lack of specific T cell activation in the presence of CXCR5-negative primary cells and CXCR5-negative T-ALL cell line Jurkat. The CXCR5-CD28 CAR-T cells of the present invention show specific IFN-γ release in response to co-culture with JeKo-1 target cells expressing CXCR5.

[0322] Example

[0323] The present invention is illustrated by the following disclosed examples. The examples provide technical support and more detailed description of potentially preferred non-limiting embodiments of the present invention.

[0324] To demonstrate the functionality and beneficial properties of the CARs described herein, the following examples should be considered:

[0325] - Co-culture of CAR-transduced human T cells with different target cell lines showed specific activation of T cells by different CXCR5+ cell lines; the readout was the release of IFN-γ from T cells as an effector cytokine.

[0326] - Cytotoxicity assays revealed selective killing of CXCR5+ cell lines; there was essentially no killing in CXCR5-negative cell lines (eg, Nalm6 and NCI-H929).

[0327] - In vivo experiments involve using a xenograft NSG mouse model to generate data on i) functionality, ii) off-target reactivity, iii) T cell memory, and iv) biosafety of adoptively transferred CAR-T cells against B-NHL cell lines. For B-NHL, the cytolytic capacity of anti-CXCR5 CAR-T cells was compared with an established anti-CD19 CAR-T cell product.

[0328] Example 1: Cloning and plasmid preparation:

[0329] CAR scFv sequences were generated using GeneArt TM(Gene Synthesis Service). Using NotI and Csil ( Figure 7 ) were subjected to restriction digestion of the synthetic sequence to release the scFv insert from the GeneArt plasmid. The retroviral vector MP71 containing the additional CAR sequence was digested with NotI and Csil to enable the separated scFv insert to be subsequently connected to the CAR construct in the vector. The digested vector was then dephosphorylated. The fragments were separated using gel electrophoresis ( Figure 8 ) and purified.

[0330] The CAR scFv sequence produced as described above was then connected to a purified vector containing another CAR sequence (50ng) at a ratio of 3: 1. The connection mixture was transformed into MACH-1. Control digestion was performed, and the micropreparation was sequenced. The construct was then re-transformed into MACH-1. The maximum amount of preparation of MP71-CXCR5-CAR plasmid was prepared.

[0331] MP71 is a single (+) stranded RNA virus. Reverse transcriptase converts the retroviral RNA-genome into a DNA copy. The DNA is integrated into the target genome at random locations as a provirus. Through cell division, the virus is stably regenerated as a provirus.

[0332] MP71 contains the following cis-regulatory elements:

[0333] The LTR (= long terminal repeat) is derived from the murine myeloproliferative sarcoma virus (MPSV) containing the promoter. The leader sequence of the vector is derived from the murine embryonic stem cell virus. The PRE (post-transcriptional regulatory element) was originally derived from the woodchuck hepatitis virus.

[0334] MP71 has been inactivated because the retroviral genes are missing and therefore no longer have the ability to replicate. The structural genes were previously introduced separately into two helper plasmids in the packaging cell line. The gag, pol and env gene products are required for transfection. Infectious viral particles are released into the cell culture supernatant and can be used to transduce PBL. High transduction rates and high expression rates can be achieved in human PBL.

[0335] Example 2: Transfection and transduction:

[0336] Day 0: Seed HEK-T (293T) cells or GalV cells in 6-well plates for virus production

[0337] Day 1: Transient 3 plasmid transfection (calcium phosphate transfection) for retrovirus production. 18 μg DNA per well was used in 250 mM Cacl2 and 150 μl H2O according to standard protocols. Cells were incubated at 37°C for 6 hours, the medium was changed, and then further incubated at 37°C for 48 hours.

[0338] Coat 24-well non-tissue culture plates with anti-huCD3 and anti-huCD28 antibodies:

[0339] Prepare a PBS solution of anti-CD3 / anti-CD28-antibodies (5 μg / ml anti-CD3, 1 μg / ml anti-CD28), 0.5 ml per well. Incubate each well with 0.5 ml of antibody solution at 37°C for 2 hours, replace with 2% sterile BSA solution (aqueous solution), incubate for 30 minutes (37°C). Remove the BSA solution and wash the wells with 2 ml PBS.

[0340] PBMCs (approximately 2.5 × 10 7 PBMCs):

[0341] Prepare 12.5 ml of Ficoll gradient medium in 2×50 ml Falcon tubes, dilute blood to 45 ml with RPMI (+100 IU / ml penicillin, streptomycin), mix, and coat with 22.5 ml of blood-medium mixture, centrifuge (20 minutes, 20°C, 1800 rpm, RZB*648, G 17.9). Discard 15 ml of the upper phase. Transfer the remaining upper phase and the middle phase containing milky white PBMCs to a new 50 ml Falcon tube, fill to 45 ml with RPMI (+100 IU / ml penicillin, streptomycin), and centrifuge. Resuspend the precipitate in 45 ml RPMI (+100 IU / ml penicillin, streptomycin), centrifuge, combine the precipitate in 10-20 ml T cell culture medium, stain one sample with trypan blue, count the cells and add a concentration of 1 to 1.5×10 6 10 cells / ml of cells (T cell culture medium (+100 IU / ml IL-2) corresponding to 400 U / ml clinical IL2) to anti-CD3, anti-CD28 coated wells. The remaining PBMCs were centrifuged, suspended in freezing medium and stored in cryovials at -80°C.

[0342] Day 3: Transduction of PBL

[0343] Viral supernatant was removed from HEK-T- or GalV cells and filtered (0.45 μm filter). Stimulated PBMCs were treated with 1.0 ml of viral supernatant. Stimulated PBLs were treated with 1 ml of viral supernatant and centrifuged in CD3 / CD28 coated wells (90 min, 32°C, 800×g). The final concentration was 100 IU / ml IL2 or 10 ng / ml IL7 and 10 ng / ml IL15, in addition to 4 μg / ml (8 μl) protamine sulfate.

[0344] Day 4: Transduction of PBL

[0345] Filter (0.45 μm) the remaining viral supernatant (4°C) and the second supernatant from HEK-T cells or GalV cells. Collect 1 ml of supernatant from PBL. Adjust the cytokine concentration to 100 IU / ml IL2 or 10 ng / ml IL7 and 10 ng / ml IL15, and 4 μg / ml (8 μl) of protamine sulfate. Centrifuge at 800 × g for 90 minutes at 32°C. 4 hours after transduction, wash PBL out of the 24-well plate into a T25 cell culture flask. Add fresh medium with IL2 or IL7 / IL15.

[0346] Day 7 to Day 13: Culture PBL and treat T cell culture medium with fresh IL2 or IL7 / IL15.

[0347] Day 13: End T cell stimulation.

[0348] The PBL cultures in the cell culture flasks were rinsed, centrifuged and the pellets were resuspended in T cell culture medium (+10 IU / ml IL2 or 1 ng / ml IL7 / IL15).

[0349] From day 15: functional assays

[0350] Example 3: In vitro functional testing of anti-CXCR5 CAR T cells

[0351] CXCR5 on human T cells after retroviral transduction Confirmation of CAR expression:

[0352] Provided evidence for folding and trafficking of CAR receptors in the context of human T cells. Demonstrated functionality of the retroviral transduction protocol.

[0353] 1) Human peripheral blood leukocytes were purified by Ficoll gradient. Cells were cultured, stimulated and transduced with retrovirus as described above.

[0354] After transduction, cells were further cultured in medium containing IL2 or IL-7 / IL15 and then analyzed for CXCR5-CAR expression.

[0355] 2) Transduction rate and viability were evaluated by flow cytometry (FACS) analysis. To detect the expression of CXCR5-CAR, cells were stained with anti-human IgG antibodies that selectively recognized the human IgG1 or IgG4 portion in the spacer region of the CAR construct. Composite staining of CD3 / CD8 T cells was performed.

[0356] The results are as follows Fig. 9 shown.

[0357] Co-culture of CAR-transduced human T cells with different target cell lines shows different CXCR5 + Characteristics of B-NHL cell lines Heterotropic T cell activation

[0358] The readout for CAR-T cell activation is the release of IFN-γ from T cells as an effector cytokine.

[0359] 1) As described in detail above, retrovirally transduced human T cells were generated. The following T cells were used: CXCR5-CD28 CAR receptor variant (H28), CXCR5-4-1BB CAR receptor variant (HBB1), CXCR5-CD28 / 4-1BB CAR receptor variant (H288BB), SP6 negative control CAR, UT = untransduced T cells.

[0360] 2) Co-culture retrovirally transduced T cells in the presence of the listed cell lines or primary cells at a 1:1 ratio for 18 to 20 hours.

[0361]

[0362] 3) After co-culture, the cell-free culture supernatant was sampled and the control level of IFN-γ was determined. The maximum release value was induced by PMA / ionomycin stimulation of effector T cells. The minimum release value was induced by T cells alone.

[0363] 4) IFN-γ release in the supernatant was measured by ELISA.

[0364] The results are as follows Fig.11 shown.

[0365] Cytotoxicity assays revealed selective killing of CXCR5-positive cell lines; in CXCR5-negative cell lines No casualties were seen

[0366] use 51 The Cr release assay quantifies the activity of cytotoxic T lymphocytes. This assay is able to measure the cytolysis of target cells.

[0367] 1) Retrovirally transduced human T cells were generated as described above. In addition to the SP6 negative control CAR and UT = untransduced T cells, a CXCR5 CAR receptor variant (H28) was used.

[0368] 2) Use 51 Cr labeled target cells

[0369] 3) CAR-T cells and labeled target cells were then co-cultured for 4 hours, and the ratio of effector cells to target cells was measured by titration:

[0370] Effect-target ratio: 80:1 0:1 20:1 10:1 5:1 2.5:1

[0377] 4) Collecting cell-free cell culture supernatant,

[0378] 5) The supernatant was transferred to a LUMA-scintillation plate and the released 51 Cr. Maximum release values ​​were determined from target cells lysed by application to LUMA plates; minimum release values ​​were determined using target cells only.

[0379] The results are as follows Fig.12 shown.

[0380] Example 4: In vivo experiments using a xenograft NSG mouse model to evaluate adoptively transferred CAR-T cells against B-NHL cell lines

[0381] To demonstrate that CAR-T cells equipped with various anti-CXCR5-CAR variants described herein have effector activity, also in situ conditions, CXCR5+ B-NHL cell lines were transplanted into NSG mice (NOD.Cg-Prkdc scid Iq tm1 Wjl / SzJ).

[0382] CXCR5+ B-NHL cell lines are:

[0383] SU-DHL4 (DLBCL), JEKO-1 (mantle cell lymphoma), JVM3 (CLL), DOHH-2 (FL), OCI-Ly7, or SC-1.

[0384] A negative control cell line was included, REH(ALL), which was not destroyed by anti-CXCR5 CAR T cells in vitro but is the target of the anti-CD19 CAR.

[0385] These mice are particularly suitable for xenotransplantation because they support the growth of human cells by providing cytokines and are devoid of T, B, and NK cells that would serve to reject the transplant. The B-NHL cell line is also stably transduced with the firefly luciferase gene, which allows for in vivo tumor cell detection by application of luciferin. The progression and distribution of injected tumors are monitored by bioluminescent imaging using the IVIS system.

[0386] Next, when the growth of tumor cells was confirmed by the increase in the intensity of the luciferase luminescence signal, about 5 to 8 days after the tumor cell inoculation, 5 × 10 5 Up to 5×10 6Human CAR T cells were administered in a titrated manner. Anti-CXCR5 CAR T cells were compared with anti-CD19 CAR and two appropriate negative controls (e.g., irrelevant SP6 control and untransduced T cell control).

[0387] The luminescence intensity was measured every 3 to 5 days, and the growth retardation / progression and disappearance of tumor cell-related signals in the animals were determined. The observation interval was 28 days. The reduction in luminescence intensity and the disappearance of tumor cell-related signals indicated that the CAR T cells had a therapeutic effect.

[0388] The above experiments used NOD.Cg-Prkdc scid Iq tm1 Wjl / SzJ (NSG) mice were treated with 5×10 5 Mice were inoculated intravenously with mantle cell lymphoma (MCL) cells transduced with the luciferase gene together with GFP ( Fig.13 ). The mice then received 3×10 6 T cells transduced with anti-CXCR5 CAR (n=4), and T cells transduced with SP6-CAR (n=3) (day 0) as negative control. The average value of bioluminescent signal was obtained from the target area covering the whole body of each mouse. Data were plotted for each group at each time point ( Fig.13 C). Essentially all animals treated with SP6 CAR developed progressive lymphomatous disease characterized by strong luminescent signals in the bone marrow of the hind limbs, thorax, and abdominal organs, whereas this was clearly not the case in the CXCR5 CAR-treated group. This provides the first preclinical in vivo demonstration that CXCR5 CAR-T cells have antitumor activity against B-NHL lymphoma entities.

[0389] Fig.14 Alternative CAR components of third generation CARs with (1) 4-1BB or CD28 as co-stimulatory components and (2) CD28 and 4-1BB as co-stimulatory components are shown, as well as their functional effects on co-culture experiments using CAR T cells and tumor cell lines. Specific activation of CAR-T cells by tumor cells bearing CXCR5 was demonstrated by the release of IFN-γ, IL-2, and TNF-α.

[0390] Although the CXCR5-CD28 (H28) CAR appears to be the most potent, two alternative CARs, CXCR5-4-1BB (HBB1) and CXCR5-CD28 / 4-1BB (H28BB), also showed significant specific activity. Fig.15 Primary cells from healthy human tissues that do not carry CXCR5 are shown (see also Fig.10Data in B) did not induce specific activity of CXCR5-CD28 CAR-T cells.

[0391] Example 5: Clinical Methods for Specific Patient Populations

[0392] In the human in vivo setting, the present invention is expected to show efficacy in B-NHL patients suffering from diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, and mantle cell lymphoma. Patients with the following characteristics will be included in the Phase I clinical study: i) patients with multidrug resistance; ii) patients who are not eligible for allogeneic stem cell transplantation; iii) patients with comorbidities that cannot receive further chemotherapy; iv) elderly patients who are intolerant to chemotherapy; v) patients who are undergoing salvage therapy after progressive disease and have failed multiple lines of other standard of care therapies, vi) patients with rapidly progressive disease after autologous stem cell transplantation, vii) patients with progressive disease after allogeneic stem cell transplantation, viiii) patients as a bridge therapy before allogeneic stem cell transplantation, and / or ix) patients who express escape variants or mutants of CD19 and / or CD20 on tumor cells, such that current antibody therapies (anti-CD20, rituximab, anti-CD19, oremertuzumab, BITE CD19 / CD3, blinatumomab) or anti-CD19 CAR therapies lose / downregulate their target structure and become ineffective.

[0393] In the human in vivo environment, the present invention is expected to show efficacy in patients with T-NHL with angioimmunoblastic T-cell lymphoma and various forms of T-cell lymphoma with leukemic dissemination, skin localization or any other organ dissemination. For these patients, there is no selective treatment except systemic chemotherapy regimens. Therefore, patients with the following characteristics will be included in the Phase I clinical study: i) patients with multidrug resistance; ii) patients who are not eligible for allogeneic stem cell transplantation; iii) patients with comorbidities that cannot accept further chemotherapy; iv) elderly patients who are intolerant to chemotherapy; v) patients who take salvage therapy after the occurrence of progressive disease, and other standard care therapies in the first or second line are ineffective, vi) patients with rapidly progressive disease after autologous stem cell transplantation, vii) patients with progressive disease after allogeneic stem cell transplantation, viii) patients with progressive disease before allogeneic stem cell transplantation as a transitional therapy, ix) patients with progressive disease who undergo first-line standard chemotherapy as a second-line therapy.

Claims

1. A chimeric antigen receptor (CAR) polypeptide comprising: i. an extracellular antigen binding domain comprising an antibody or antibody fragment that binds to a CXC chemokine receptor type 5 (CXCR5) protein, ii. transmembrane domain, and iii. Intracellular domain, in, The antigen binding domain comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises: - the heavy chain complementarity determining region 1 (H-CDR1) according to SEQ ID NO. 1 (GFTFSTSG), - the heavy chain complementarity determining region 2 (H-CDR2) according to SEQ ID NO. 2 (ISSSSGFV), and - the heavy chain complementarity determining region 3 (H-CDR3) according to SEQ ID NO. 3 (ARSEAAF), The VL includes: - light chain complementary determining region 1 (L-CDR1) according to SEQ ID NO. 4 (KSRLSRMGITP), - the light chain complementarity determining region 2 (L-CDR2) according to SEQ ID NO. 5 (RMS), and - Light chain complementarity determining region 3 (L-CDR3) according to SEQ ID NO. 6 (AQFLEYPPT).

2. The chimeric antigen receptor (CAR) polypeptide of claim 1, comprising: A VH domain having at least 90% sequence identity with SEQ ID NO. 7 as follows: EVQLVESGGGLVQPGX1SLX2LSCX3ASGFTFSTSGMX4WFRQAPGKGLX5WVX6YISSSSGFVYADX7VKGRFTISRDNAQNX8LYLQX9NSLX10X11EDTAX12YYCARSEAAFWGQGTLVTVSS, in, X1: G or K, X2: R or K; X3: A or S; X4: N or H; X5: E or D; X6: S or A; X7: S or A; X8: S or T; X9: M or L; X10: R or K, X11: A or S; X12: V or I; and a VL domain, wherein the VL domain has at least 90% sequence identity with SEQ ID NO. 8 as follows: DIVLTQX1PRSX2PVTPGEX3ASISCRSX4KSRLSRMGITPLNWYLQKPGX5SPQLLIYRMSNX6ASGVPDRFSGSGSX7TDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK, Among them, X1: S or A; X2: L or V; X3: P or S; X4: S or N; X5: Q or K; X6: R or L; X7: G or E, Wherein, the VH domain comprises CDR sequences of H-CDR1 according to SEQ ID NO.1, H-CDR2 according to SEQ ID NO.2 and H-CDR3 according to SEQ ID NO.3; and the VL domain comprises CDR sequences of L-CDR1 according to SEQ ID NO.4, L-CDR2 according to SEQ ID NO.5 and L-CDR3 according to SEQ ID NO.

6.

3. The chimeric antigen receptor (CAR) polypeptide of claim 1, comprising: According to the VH domain of SEQ ID NO.9 as follows: EVQLVESGGGLVQPGGSLRLSCAASGFTFSTSGMNWFRQAPGKGLEWVSYISSSSGFVYADSVKGRFTISRDNAQNSLYLQMNSLRAEDTAVYYCARSEAAFWGQGTLVTVSS, or According to the VH domain of SEQ ID NO.10 as follows: EVQLVESGGGLVQPGKSLKLSCSASGFTFSTSGMHWFRQAPGKGLDWVAYISSSSGFVYADAVKGRFTISRDNAQNTLYLQLNSLKSEDTAIYYCARSEAAFWGQGTLVTVSS; and a VL domain according to SEQ ID NO. 11 as follows: DIVLTQSPRSLPVTPGEPASISCRSSKSRLSRMGITPLNWYLQKPGQSPQLLIYRMSNRASGVPDRFSGSGSGTDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK, or According to the VL domain of SEQ ID NO. 12 as follows: DIVLTQAPRSVSVTPGESASISCRSNKSRLSRMGITPLNWYLQKPGKSPQLLIYRMSNLASGVPDRFSGSGSETDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK.

4. The chimeric antigen receptor (CAR) polypeptide according to claim 1, wherein When the CAR is expressed in a genetically modified immune cell, the immune cell binds to CXCR5 on the surface of a cell expressing CXCR5 and is activated, thereby inducing cytotoxic activity against the cell expressing CXCR5.

5. The chimeric antigen receptor (CAR) polypeptide according to claim 4, wherein The cells expressing CXCR5 are DOHH-2, OCI-Ly7, SU-DHL4, JeKo-1, JVM-3, MEC-1 and / or SC-1 cell lines.

6. The chimeric antigen receptor (CAR) polypeptide according to claim 4, wherein The genetically modified immune cells are T lymphocytes.

7. The chimeric antigen receptor (CAR) polypeptide according to claim 1: - wherein the extracellular antigen binding domain comprises a linker polypeptide located between the VH domain and the VL domain, wherein the linker is selected from the group consisting of Whitlow according to SEQ ID NO. 13, or a Gly-Ser linker according to SEQ ID NO. 14; and / or - further comprising a spacer polypeptide located between the extracellular antigen binding domain and the transmembrane domain, wherein the spacer is selected from: a. According to the IgG1 spacer region of SEQ ID NO.15, b. IgG1Δ spacer according to SEQ ID NO.16, c. the IgG4 spacer according to SEQ ID NO.17, d. an IgG4 spacer according to SEQ ID NO.18, or e. an IgG4 spacer according to SEQ ID NO.19; and / or -in, The transmembrane domain is selected from the CD8α domain according to SEQ ID NO. 20 or the CD28 domain according to SEQ ID NO. 21; and / or - wherein the intracellular domain comprises a co-stimulatory domain, the co-stimulatory domain being selected from the co-stimulatory domain of 4-1BB according to SEQ ID NO.22, or the co-stimulatory domain of CD28 according to SEQ ID NO.23, or a co-stimulatory domain comprising both the co-stimulatory domain of 4-1BB according to SEQ ID NO.22 and the co-stimulatory domain of CD28 according to SEQ ID NO.23 arranged adjacently; and / or - additionally comprises a signaling domain, wherein the signaling domain is the CD3zeta signaling domain according to SEQ ID NO.

24.

8. The chimeric antigen receptor (CAR) polypeptide of claim 1, comprising or consisting of any one of SEQ ID NO. 25, 26, 27, 28 or 29.

9. A nucleic acid molecule encoding the chimeric antigen receptor (CAR) polypeptide according to claim 1.

10. The nucleic acid molecule according to claim 9, comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular domain, wherein: The extracellular antigen binding domain is encoded by at least one sequence of SEQ ID NO. 37, 53 or 38 and at least one sequence of SEQ ID NO. 39, 54 or 40, or the nucleic acid molecule comprises a sequence according to SEQ ID NO. 31, 32, 33, 34 or 35.

11. A genetically modified immune cell comprising the nucleic acid molecule according to claim 9 or 10 and / or expressing the CAR according to claim 1.

12. The genetically modified immune cell according to claim 11, wherein The immune cells are selected from the group consisting of T lymphocytes and NK cells.

13. The genetically modified immune cell according to claim 12, wherein The T lymphocytes are cytotoxic T lymphocytes.

14. Use of a genetically modified immune cell according to any one of claims 11 to 13 in the preparation of a preparation for treating a medical condition associated with the presence of pathogenic cells expressing CXCR5, wherein The medical condition is: - B-cell non-Hodgkin lymphoma (B-NHL); - T-cell non-Hodgkin's lymphoma; - a lymphoproliferative disorder of B-cell origin selected from the group consisting of acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL); - a lymphoproliferative disorder of T-cell origin selected from the group consisting of angioimmunoblastic T-cell lymphoma, cutaneous T-cell lymphoma and T-cell lymphoma with leukemic disseminated spread; or - an autoantibody-dependent autoimmune disease selected from systemic lupus erythematosus (SLE) or rheumatoid arthritis.

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