Chimeric Antigen Receptor (CAR) Molecule Isolated and Its Use, Humanized Anti-CD19 Binding Domain, and Method for Generating a Population of RNA-Engineered Cells

Abstract

Isolated nucleic acid molecules, polypeptide and chimeric antigen receptor (CAR), humanized anti-CD19 binding domain, vector, cells and method of producing them, use of said molecules, binding domain, vector and cells, method for generating a population of RNA-engineered cells, and method for producing an RNA transcript in vitro encoding a CD19 CAR. The present invention relates to compositions and methods for treating diseases associated with CD19 expression. The invention also relates to the chimeric antigen receptor (CAR) specific for CD19, to vectors encoding it, and to recombinant T cells comprising the CD19 CAR. The invention also includes methods for delivering a genetically modified T cell expressing a CAR comprising a CD19-binding domain.

Description

Chimeric Antigen Receptor (CAR) Molecule Isolated and Its Use, Humanized Anti-CD19 Binding Domain, and Method for Generating a Population of RNA-Engineered Cells

[0001] This patent application claims priority to this US Patent Application Serial No. 61 / 802 629, filed March 16, 2013, and US Patent Application Serial No. 61 / 838 537, filed June 24, 2013, the contents of which are incorporated herein in their entirety by reference in this patent application. Sequence listing

[0002] This patent application contains a Listing of The sequence was submitted electronically in ASCII format and is incorporated herein, in its entirety, by reference in this patent application. The aforementioned ASCII copy, created on March 14, 2014, is named N2067-7002WO_SL.txt and is 228,415 bytes in size. Field of invention

[0003] The present invention relates generally to the use of T cells constructed to express a Chimeric Antigen Receptor (CAR) in order to treat a disease associated with the expression of the Cluster of Differentiation protein 19 (CD19). Background of the invention

[0004] Many patients have B-cell-related malignancies that are incurable with standard therapy. Furthermore, traditional treatment options often carry serious side effects. Immunotherapy has been attempted against cancer; however, several obstacles make clinical efficacy a very difficult goal to achieve. Although hundreds have been identified, the so-called tumor antigens generally derive from the organism itself and are therefore of weak immunogenicity. In addition, tumors make use of various... Petition 870260065699, dated 03 / 07 / 2026, page 9 / 532 2 / 250 mechanisms that make them hostile to the introduction and spread of immune attacks.

[0005] Recent developments using autologous T-cell therapy modified to express a chimeric antigen receptor (CAR) (CART), which relies on redirecting T cells to a suitable molecule on the cell surface, such as B-cell malignancies, show promising results in harnessing the power of the immune system to treat B-cell malignancies and other types of cancer (see, for example, Sadelain et al., Cancer Discovery 3:388-398 (2013)). Clinical results from murine-derived CART19 cells (i.e., CTL019) have shown promise in establishing complete remissions for patients suffering from chronic lymphocytic leukemia (CLL) as well as childhood acute lymphoblastic leukemia (ALL) (see, for example, Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368:1509-1518 (2013)).In addition to the ability conferred on the chimeric antigen receptor in genetically modified T cells to recognize and destroy target cells, for T cell therapy to be therapeutically successful, it must also confer the ability to proliferate and persist over time, and to monitor the escape of leukemic cells. The variable quality of T cells, whether resulting from anergy, suppression, or exhaustion, will affect the performance of CAR-transformed T cells, but over which competent physicians have limited control at this time. For them to be effective, the ability to proliferate in response to the CAR antigen must persist and be maintained by CAR-transformed T cells from patients. It has been demonstrated that T cells from patients with ALL achieve this performance with CART19 comprising a murine scFv (see, for example, Grupp et al.). Petition 870260065699, dated 03 / 07 / 2026, page 10 / 532 3 / 250 al., NEJM 368:1509-1518 (2013)). Summary of the invention

[0006] The invention addresses controlling an immune response in patients by providing optimized and humanized antibody fragments (e.g., scFv) that bind to the Differentiation Group 19 (CD19) protein integrated into a constructed Chimeric Antigen Receptor (CAR) that will not elicit an immune response in patients, is safe for long-term use, and maintains or has better clinical efficacy when compared to known CART therapy for the treatment of B-cell-derived cancers. The invention further pertains to the use of T cells constructed to express a humanized antibody fragment that binds to CD19 integrated into a CAR to treat a hematologic cancer associated with CD19 expression (OMIM Accession No.: 107265, Swiss Prot. Accession No.: P15391).

[0007] Thus, in one aspect, the invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment that includes an anti-CD19 humanized binding domain, a transmembrane domain, and an intracellular signaling domain (for example, an intracellular signaling domain comprising a co-stimulatory domain and / or a primary signaling domain). In one embodiment, the CAR comprises an antibody or antibody fragment that includes an anti-CD19 humanized binding domain described herein, a transmembrane domain described herein, and an intracellular signaling domain described herein (for example, an intracellular signaling domain comprising a co-stimulatory domain and / or a primary signaling domain).

[0008] In one embodiment, the encoded humanized antiCD19 binding domain comprises one or more of (e.g., all) Petition 870260065699, dated 03 / 07 / 2026, p. 11 / 532 4 / 250 as three) the complementarity-determining region 1 of the light chain (LC CDR1), the complementarity-determining region 2 of the light chain (LC CDR2), and the complementarity-determining region 3 of the light chain (LC CDR3) of a humanized anti-CD19 binding domain described herein and / or one or more (e.g., all three) between the complementarity-determining region 1 of the heavy chain (HC CDR1), the complementarity-determining region 2 of the heavy chain (HC CDR2), and the complementarity-determining region 3 of the heavy chain (HC CDR3) of a humanized anti-CD19 binding domain described herein, for example, a humanized anti-CD19 binding domain comprising one or more, e.g., all three LC CDRs, and one or more, e.g., all three HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2.In one embodiment, the encoded humanized anti-CD19 binding domain comprises one or more (e.g., all three) of the heavy chain complementarity-determining region 1 (HC CDR1), the heavy chain complementarity-determining region 2 (HC CDR2), and the heavy chain complementarity-determining region 3 (HC CDR3) of an anti-CD19 humanized binding domain described herein; for example, the encoded humanized anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1, an HC CDR2, and an HC CDR3 described herein. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the encoded variable light chain region comprises one, two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the encoded variable light chain region has a modification (e.g.,...). Petition 870260065699, dated 03 / 07 / 2026, p. 12 / 532 5 / 250 substitution, for example, a substitution of one or more amino acids found at the corresponding position in the variable region of the light chain of SEQ ID NO: 58, for example, a substitution at one or more of the positions 71 and 87). In one embodiment, the encoded variable region of the heavy chain comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the encoded variable region of the heavy chain has a modification (e.g., substitution, for example, a substitution of one or more amino acids found at the corresponding position in the variable region of the heavy chain of SEQ ID NO: 58, for example, a substitution at one or more of the positions 71, 73, and 78). In one embodiment, the encoded humanized anti-CD19 binding domain comprises a variable region of the heavy chain described herein (e.g., in Table 3) and / or a humanized variable region of the heavy chain described herein (e.g., in Table 3).In one embodiment, the encoded humanized anti-CD19 binding domain comprises a humanized heavy chain variable region described herein (e.g., in Table 3), for example, at least two humanized heavy chain variable regions described herein (e.g., in Table 3). In one embodiment, the anti-CD19 binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence from Table 3. In one embodiment, the anti-CD19 binding domain (e.g., a scFv) comprises: a light chain variable region comprising an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence from a light chain variable region provided in Table 3, or a sequence with 95-99% identity to an amino acid sequence from Table 3; and / or a heavy chain variable region. Petition 870260065699, dated 03 / 07 / 2026, p. 13 / 532 6 / 250 comprising an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence from a variable region of the heavy chain provided in Table 3, or a sequence with 95-99% identity with respect to an amino acid sequence from Table 3. In one embodiment, the encoded humanized anti-CD19 binding domain comprises a sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or a sequence with 95-99% identity with these sequences.In one embodiment, the nucleic acid sequence encoding the anti-CD19 humanized binding domain comprises a sequence selected from a group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, or a sequence with 95-99% identity to these. In one embodiment, the encoded anti-CD19 humanized binding domain is an scFv, and a variable region of the light chain comprising an amino acid sequence described herein, for example, in Table 3, is linked to a variable region of the heavy chain comprising an amino acid sequence described herein, for example, in Table 3, via a linker, for example, a linker described herein. In one embodiment, the encoded humanized anti-CD19 linking domain includes a linker (Gly4-Ser)n, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO:53).The variable region of the light chain and the variable region of the heavy chain of a scFv can be, for example, in any of the following orientations: variable region of the light chain-linker-variable region of the heavy chain. Petition 870260065699, dated 03 / 07 / 2026, page 14 / 532 7 / 250 or variable region of the heavy chain-linker-variable region of the light chain.

[0009] In one embodiment, the encoded transmembrane domain is a transmembrane domain of a protein selected from the alpha, beta, or zeta chain group of the T-cell receptor CD27, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the encoded transmembrane domain comprises a sequence of SEQ ID NO: 15. In one embodiment, the encoded transmembrane domain comprises an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO:15, or a sequence with 95-99% identity with respect to an amino acid sequence of SEQ ID NO:15. In one embodiment, the nucleic acid sequence encoding the transmembrane domain comprises a sequence with SEQ ID NO:56, or a sequence with 95-99% identity thereof.

[0010] In one embodiment, the encoded anti-CD19 binding domain is connected to the transmembrane domain by a hinge region, for example, a hinge region described herein. In one embodiment, the encoded hinge region comprises SEQ ID NO:14, SEQ ID NO:45, or SEQ ID NO:47, or a sequence with 95-99% identity thereof. In one embodiment, a nucleic acid sequence encoding the hinge region comprises a sequence of SEQ ID NO:55, SEQ ID NO:46, or SEQ ID NO:48, or a sequence with 95-99% identity thereof.

[0011] In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In one embodiment, the costimulatory domain is a Petition 870260065699, dated 03 / 07 / 2026, page 15 / 532 8 / 250 functional signaling domain obtained from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137). In one embodiment, the encoded costimulatory domain comprises a sequence with SEQ ID NO:16. In another embodiment, the encoded costimulatory domain comprises an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions) of an amino acid sequence with SEQ ID NO:16, or a sequence with 95-99% identity to an amino acid sequence with SEQ ID NO:16. In another embodiment, the nucleic acid sequence encoding the costimulatory domain comprises a sequence with SEQ ID NO:60, or a sequence with 95-99% identity to that sequence.In one embodiment, the isolated nucleic acid molecule further comprises a coding sequence for an intracellular signaling domain, for example, an intracellular signaling domain described herein. In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 zeta. In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of CD27 and / or a functional signaling domain of CD3 zeta. In one embodiment, the encoded intracellular signaling domain comprises the sequence SEQ ID NO: 16 or SEQ ID NO: 51 and / or the sequence SEQ ID NO: 17 or SEQ ID NO: 43.In one embodiment, the intracellular signaling domain comprises an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO:16 or SEQ ID NO:51 and / or an amino acid sequence of... Petition 870260065699, dated 03 / 07 / 2026, p. 16 / 532 9 / 250 SEQ ID NO:17 or SEQ ID NO:43, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO:16 or SEQ ID NO:51 and / or an amino acid sequence of SEQ ID NO:17 or SEQ ID NO:43. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO:16 or SEQ ID NO:51 and the sequence of SEQ ID NO:17 or SEQ ID NO:43, wherein the sequences comprising the intracellular signaling domain are expressed in the same reading frame and as a single polypeptide chain. In another embodiment, the nucleic acid sequence encoding the intracellular signaling domain comprises a sequence of SEQ ID NO:60, or a sequence with 95-99% identity to it, and / or a sequence of SEQ ID NO:101 or SEQ ID NO:44, or a sequence with 95-99% identity to it.In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain comprises a sequence with SEQ ID NO:52, or a sequence with 95-99% identity thereof, and / or a sequence with SEQ ID NO:101 or SEQ D NO:44, or a sequence with 95-99% identity thereof.

[0012] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a constructed CAR, comprising a leader sequence, for example, the leader sequence described herein, for example, of SEQ ID NO:13; a humanized anti-CD19 binding domain described herein, for example, a humanized anti-CD19 binding domain comprising an LC CDR1, an LC CDR2, an LC CDR3, an HC CDR1, an HC CDR2 and an HC CDR3 described herein, for example, a humanized anti-CD19 binding domain described in Table 3, or a sequence with 95-99% identity thereof; a linkage region described herein, for example, of SEQ ID NO:14 or SEQ ID NO:45; a transmembrane domain described herein, for example, a transmembrane domain comprising the Petition 870260065699, dated 03 / 07 / 2026, p. 17 / 532 10 / 250 SEQ ID NO:15; and an intracellular signaling domain, for example, an intracellular signaling domain described herein. In one embodiment, the encoded intracellular signaling domain comprises a co-stimulatory domain, for example, a co-stimulatory domain described herein, for example, a 4-1BB co-stimulatory domain having a sequence of SEQ ID NO:16 or SEQ ID NO:51, and / or a primary signaling domain, for example, a primary signaling domain as described herein, for example, a CD3 zeta stimulatory domain having a sequence of SEQ ID NO:17 or SEQ ID NO:43. In one embodiment, the isolated nucleic acid molecule encoding the constructed CAR includes a leader sequence encoded by the nucleic acid sequence of SEQ ID NO:54, or a sequence with 95-99% identity with respect to that sequence.In one embodiment, the constructed CAR-encoding isolated nucleic acid molecule includes a sequence of the anti-CD19 humanized binding domain encoded by the nucleic acid sequence SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72, or a sequence with 95-99% identity to them. In another embodiment, the constructed CAR-encoding isolated nucleic acid molecule includes a transmembrane sequence encoded by the nucleic acid sequence SEQ ID NO:56, or a sequence with 95-99% identity to it.In one embodiment, the isolated nucleic acid molecule encoding the constructed CAR includes a sequence of the intracellular signaling domain encoded by the nucleic acid sequence SEQ ID NO:60, or a sequence with 95-99% identity to this, and / or a nucleic acid sequence SEQ ID NO:101 or SEQ ID NO:44, or a sequence with 95-99% identity to these. Petition 870260065699, dated 03 / 07 / 2026, p. 18 / 532 11 / 250

[0013] In one embodiment, the isolated nucleic acid molecule comprises (for example, consists of) a nucleic acid encoding an amino acid sequence of CAR from SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42, or an amino acid sequence containing at least one, two, three, four, five, 10, 15, 20 or 30 modifications (for example, substitutions), but not more than 60, 50 or 40 modifications (for example, substitutions) of an identical amino acid sequence or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with respect to an amino acid sequence of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42.

[0014] In one embodiment, the isolated nucleic acid molecule comprises (for example, consists of) a nucleic acid sequence with SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96 or a nucleic acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with respect to a nucleic acid sequence with SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96.

[0015] In one aspect, the invention relates to an isolated nucleic acid molecule encoding a humanized anti-CD19 binding domain, wherein the anti-CD19 binding domain comprises one or more (e.g., all three) between the complementarity-determining region 1 of the light chain (LC CDR1), the Petition 870260065699, dated 03 / 07 / 2026, p. 19 / 53212 / 250 the complementarity-determining region 2 of the light chain (LC CDR2) and the complementarity-determining region 3 of the light chain (LC CDR3) of an anti-CD19 binding domain described herein, and one or more (e.g., all three) between the complementarity-determining region 1 of the heavy chain (HC CDR1), the complementarity-determining region 2 of the heavy chain (HC CDR2), and the complementarity-determining region 3 of the heavy chain (HC CDR3) of an anti-CD19 binding domain described herein, for example, a humanized anti-CD19 binding domain comprising one or more, e.g., all three, LC CDRs, and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the variable light chain region comprises one, two, three, or all four framework regions of the VK3_L25 germline sequence.In one embodiment, the variable region of the light chain has a modification (e.g., substitution, for example, a substitution of one or more amino acids found at the corresponding position in the variable region of the murine light chain of SEQ ID NO: 58, for example, a substitution at one or more of positions 71 and 87). In one embodiment, the variable region of the heavy chain comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the variable region of the heavy chain has a modification (e.g., substitution, for example, a substitution of one or more amino acids found at the corresponding position in the variable region of the murine heavy chain of SEQ ID NO: 58, for example, a substitution at one or more of positions 71, 73, and 78).In one embodiment, the encoded humanized anti-CD19 binding domain comprises a variable region of the light chain described herein (e.g., in SEQ ID NO:1,2,3,4,5,6,7,8,9,10,11, or12) and / or a... Petition 870260065699, dated 03 / 07 / 2026, page 20 / 532 13 / 250 variable region of the heavy chain described herein (e.g., in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12). In one embodiment, the encoded humanized anti-CD19 binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence represented in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In another embodiment, the humanized anti-CD19 binding domain (e.g., a scFv) comprises: a variable light chain region comprising an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence from a variable light chain region provided in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a sequence with 95% identity to an amino acid sequence from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11 or 12; and / or a variable region of the heavy chain comprising an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence from a variable region of the heavy chain provided in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a sequence with 95-99% identity with respect to an amino acid sequence from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one embodiment, the humanized anti-CD19 binding domain comprises a sequence selected from a group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a sequence with 95-99% identity of these. In one embodiment, the nucleic acid sequence encoding the anti-CD19 humanized binding domain comprises a sequence selected from a group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,SEQ ID NO:65, SEQ ID, Petition 870260065699, dated 03 / 07 / 2026, p. 21 / 532 14 / 250 NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, or a sequence with 95-99% identity of these.

[0016] In another aspect, the invention relates to an isolated polypeptide molecule encoded by a nucleic acid sequence. In one embodiment, the isolated polypeptide molecule comprises a sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42. In one embodiment, the isolated polypeptide comprises a sequence of SEQ ID NO:31. In one embodiment, the isolated polypeptide comprises a sequence of SEQ ID NO:32. In one embodiment, the isolated polypeptide molecule comprises a sequence of SEQ ID NO:35. In one embodiment, the isolated polypeptide molecule comprises a sequence of SEQ ID NO:36. In one embodiment, the isolated polypeptide molecule comprises a sequence with SEQ ID NO:37.

[0017] In another aspect, the invention relates to an isolated chimeric antigen receptor (CAR) molecule comprising a humanized anti-CD19 binding domain (e.g., a humanized antibody or antibody fragment that specifically binds to CD19), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a co-stimulatory domain and / or a primary signaling domain). In one embodiment, the CAR comprises an antibody or antibody fragment that includes a humanized anti-CD19 binding domain described herein (e.g., a humanized antibody or antibody fragment that specifically binds to CD19 as described herein), a transmembrane domain described herein, and an intracellular signaling domain as described herein. Petition 870260065699, dated 03 / 07 / 2026, page 22 / 532 15 / 250 (for example, an intracellular signaling domain comprising a co-stimulatory domain and / or a primary signaling domain, as described herein).

[0018] In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) between the complementarity-determining region 1 of the light chain (LC CDR1), the complementarity-determining region 2 of the light chain (LC CDR2), and the complementarity-determining region 3 of the light chain (LC CDR3) of a humanized anti-CD19 binding domain described herein, and one or more (e.g., all three) between the complementarity-determining region 1 of the heavy chain (HC CDR1), the complementarity-determining region 2 of the heavy chain (HC CDR2), and the complementarity-determining region 3 of the heavy chain (HC CDR3) of a humanized anti-CD19 binding domain described herein, for example, a humanized anti-CD19 binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2.In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) of the heavy chain complementarity-determining region 1 (HC CDR1), the heavy chain complementarity-determining region 2 (HC CDR2), and the heavy chain complementarity-determining region 3 (HC CDR3) of a humanized anti-CD19 binding domain described herein; for example, the humanized anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1, an HC CDR2, and an HC CDR3 described herein. In one embodiment, the humanized anti-CD19 binding domain comprises at least an HC CDR2. In one embodiment, the variable light chain region comprises one... Petition 870260065699, dated 03 / 07 / 2026, page 23 / 532 16 / 250 two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the variable region of the light chain has a modification (e.g., substitution, for example, a substitution of one or more amino acids found at the corresponding position in the murine light chain variable region of SEQ ID NO: 58, for example, a substitution at one or more of positions 71 and 87). In one embodiment, the variable region of the heavy chain comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the variable region of the heavy chain has a modification (e.g., substitution, for example, a substitution of one or more amino acids found at the corresponding position in the murine heavy chain variable region of SEQ ID NO: 58, for example, a substitution at one or more of positions 71, 73, and 78).In one embodiment, the humanized anti-CD19 binding domain comprises a variable region of the light chain described herein (e.g., in Table 3) and / or a variable region of the heavy chain described herein (e.g., in Table 3). In another embodiment, the humanized anti-CD19 binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence from Table 3.In one embodiment, the humanized anti-CD19 binding domain (e.g., an scFv) comprises: a variable light chain region comprising an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence from a variable light chain region provided in Table 3, or a sequence with 95-99% identity to an amino acid sequence from Table 3; and / or a variable heavy chain region comprising an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions). Petition 870260065699, dated 03 / 07 / 2026, page 24 / 532 17 / 250 example, substitutions), but no more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence from a variable region of the heavy chain provided in Table 3, or a sequence with 95-99% identity with respect to an amino acid sequence from Table 3. In one embodiment, the humanized anti-CD19 binding domain comprises a sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, or a sequence with 95-99% identity of these.In one embodiment, the humanized anti-CD19 binding domain is an scFv, and a variable region of the light chain comprising an amino acid sequence described herein, for example, in Table 3, is linked to a variable region of the heavy chain comprising an amino acid sequence described herein, for example, in Table 3, via a linker, for example, a linker described herein. In one embodiment, the humanized anti-CD19 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The variable region of the light chain and the variable region of the heavy chain of an scFv may be, for example, in any of the following orientations: variable region of the light chain-linker-variable region of the heavy chain or variable region of the heavy chain-linker-variable region of the light chain.

[0019] In one embodiment, the isolated CAR molecule comprises a transmembrane domain of a protein selected from the alpha, beta, or zeta chain group of the T-cell receptor CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain comprises a sequence with SEQ ID NO: 15. In one embodiment, the domain Petition 870260065699, dated 03 / 07 / 2026, p. 25 / 532 18 / 250 transmembrane comprises an amino acid sequence containing at least one, two or three modifications (e.g., substitutions), but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 15, or a sequence with 95-99% identity with respect to an amino acid sequence of SEQ ID NO: 15.

[0020] In one embodiment, the humanized antiCD19 binding domain is connected to the transmembrane domain by a hinge region, for example, a hinge region described herein. In one embodiment, the encoded hinge region comprises SEQ ID NO:14 or SEQ ID NO:45, or a sequence with 95-99% identity to these.

[0021] In one embodiment, the isolated CAR molecule further comprises a coding sequence for a costimulatory domain, for example, a costimulatory domain described herein. In one embodiment, the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA1 (CD11a / CD18) and 4-1BB (CD137). In one embodiment, the costimulatory domain comprises a sequence with SEQ ID NO: 16. In one embodiment, the costimulatory domain comprises an equal sequence with SEQ ID NO: 51.In one embodiment, the costimulatory domain comprises an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions) of an amino acid sequence with SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence with 95-99% identity with respect to an amino acid sequence with SEQ ID NO: 16 or SEQ ID NO: 51.

[0022] In one embodiment, the isolated CAR molecule Petition 870260065699, dated 03 / 07 / 2026, p. 26 / 532 19 / 250 further comprises a coding sequence for an intracellular signaling domain, for example, an intracellular signaling domain described herein. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 zeta. In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 16 and / or the sequence SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 16 and / or the sequence SEQ ID NO: 43. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of CD27 and / or a functional signaling domain of CD3 zeta. In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 51 and / or the sequence SEQ ID NO: 17.In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO:51 and / or the sequence SEQ ID NO:43. In another embodiment, the intracellular signaling domain comprises an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions) of an amino acid sequence from SEQ ID NO:16 or SEQ ID NO:51 and / or an amino acid sequence from SEQ ID NO:17 or SEQ ID NO:43, or a sequence with 95-99% identity with respect to an amino acid sequence from SEQ ID NO:16 or SEQ ID NO:51 and / or an amino acid sequence from SEQ ID NO:17 or SEQ ID NO:43.In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO:16 or SEQ ID NO:51 and the sequence SEQ ID NO:17 or SEQ ID NO:43, wherein the sequences comprising the intracellular signaling domain are expressed in the same reading frame and as a single polypeptide chain. Petition 870260065699, dated 03 / 07 / 2026, page 27 / 532 20 / 250

[0023] In one embodiment, the isolated CAR molecule further comprises a leader sequence, for example, a leader sequence described herein. In one embodiment, the leader sequence comprises an amino acid sequence of SEQ ID NO: 13, or a sequence with 95-99% identity with respect to an amino acid sequence of SEQ ID NO:13.

[0024] In another aspect, the invention relates to an isolated CAR molecule comprising a leader sequence, for example, a leader sequence described herein, for example, a leader sequence of SEQ ID NO: 13 or having 95-99% identity thereof; a humanized anti-CD19 binding domain described herein, for example, a humanized anti-CD19 binding domain comprising an LC CDR1, an LC CDR2, an LC CDR3, an HC CDR1, an HC CDR2 and an HC CDR3 described herein, for example, a humanized anti-CD19 binding domain described in Table 3, or a sequence with 95-99% identity thereof; a linkage region, for example, a linkage region described herein, for example, a linkage region of SEQ ID NO: 14 or having 95-99% identity thereof; a transmembrane domain, for example, a transmembrane domain described here, for example, a transmembrane domain having a sequence with SEQ ID NO:15 or a sequence having 95-99% identity to this;an intracellular signaling domain, for example, an intracellular signaling domain described herein (for example, an intracellular signaling domain comprising a co-stimulatory domain and / or a primary signaling domain). In one embodiment, the intracellular signaling domain comprises a co-stimulatory domain, for example, a co-stimulatory domain described herein, for example, a 4-1BB co-stimulatory domain having a sequence of SEQ ID NO:16 or SEQ ID NO:51, or having 95-99% identity thereof and / or a primary signaling domain, for example; Petition 870260065699, dated 03 / 07 / 2026, page 28 / 532 21 / 250 example, a primary signaling domain, described here, for example, a CD3 zeta stimulatory domain having a sequence with SEQ ID NO:17 or SEQ ID NO:43 or having 95-99% identity of these.

[0025] In one embodiment, the isolated CAR molecule comprises (for example, consists of) an amino acid sequence of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42, or an amino acid sequence containing at least one, two, three, four, five, 10, 15, 20 or 30 modifications (e.g., substitutions), but not more than 60, 50 or 40 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with respect to an amino acid sequence SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41 or SEQ ID NO:42.,

[0026] In one aspect, the invention relates to a humanized anti-CD19 binding domain comprising one or more (e.g., all three) between the complementarity-determining region 1 of the light chain (LC CDR1), the complementarity-determining region 2 of the light chain (LC CDR2), and the complementarity-determining region 3 of the light chain (LC CDR3) of an anti-CD19 binding domain described herein, and one or more (e.g., all three) between the complementarity-determining region 1 of the heavy chain (HC CDR1), the region Petition 870260065699, dated 03 / 07 / 2026, page 29 / 532 22 / 250 heavy chain complementarity determinant 2 (HC CDR2) and heavy chain complementarity determinant 3 (HC CDR3) regions of a humanized anti-CD19 binding domain described herein, for example, of a humanized anti-CD19 binding domain comprising one or more, for example, all three, LC CDRs and one or more, for example, all three HC CDRs. In one embodiment, the humanized anti-CD19 binding domain contains at least HC CDR2. In one embodiment, the light chain variable region comprises one, two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the light chain variable region has a modification (e.g., substitution, for example, a substitution of one or more amino acids found at the corresponding position in the murine light chain variable region of SEQ ID NO: 58, for example, a substitution at one or more of the positions 71 and 87).In one embodiment, the variable region of the heavy chain comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In another embodiment, the variable region of the heavy chain has a modification (e.g., substitution, for example, a substitution of one or more amino acids found at the corresponding position in the variable region of the heavy chain of SEQ ID NO: 58, for example, a substitution at one or more of the positions 71, 73, and 78).In one embodiment, the humanized anti-CD19 binding domain comprises a variable region of the light chain described herein (e.g., at SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12) and / or a variable region of the heavy chain described herein (e.g., at SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12). Petition 870260065699, dated 03 / 07 / 2026, page 30 / 532 23 / 250 (SEQ ID NO:12). In one embodiment, the humanized anti-CD19 binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence at SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12. In one embodiment, the humanized anti-CD19 binding domain (e.g., an scFv) comprises: a variable light chain region comprising an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence from a variable light chain region provided in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or a sequence with 95-99% identity to an amino acid sequence in SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12; and / or a variable region of the heavy chain comprising an amino acid sequence containing at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence from a variable region of the heavy chain given in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or a sequence with 95% identity to an amino acid sequence in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12., Petition 870260065699, dated 03 / 07 / 2026, page 31 / 532 24 / 250

[0027] In another aspect, the invention relates to a vector comprising a nucleic acid sequence encoding a CAR. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector or retroviral vector.

[0028] In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further comprises a promoter. In one embodiment, the promoter is an EF-1 promoter. In one embodiment, the EF-1 promoter comprises a sequence with SEQ ID NO: 100.

[0029] In one embodiment, the vector is an in vitro transcribed vector, for example, a vector that transcribes RNA from a nucleic acid molecule as described herein. In one embodiment, the nucleic acid sequence in the vector further comprises a poly(A) tail, for example, a poly(A) tail described herein, for example, comprising 150 adenosine bases (SEQ ID NO:104). In one embodiment, the nucleic acid sequence in the vector further comprises a 3'UTR, for example, a 3'UTR described herein, for example, comprising at least one repeat of a 3'UTR derived from human beta-globulin. In one embodiment, the nucleic acid sequence in the vector further comprises a promoter, for example, a T2A promoter.

[0030] In another aspect, the invention relates to a cell comprising the vector. In one embodiment, the cell is a human T cell. In one embodiment, the cell is a cell described herein, for example, a human T cell described herein. In one embodiment, the human T cell is a CD8+ T cell.

[0031] In another embodiment, the CAR-expressing cell described herein may express yet another agent, for example, an agent that enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Examples of inhibitory molecules include PD1, Petition 870260065699, dated 03 / 07 / 2026, page 32 / 532 25 / 250 PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, for example, an inhibitory molecule associated with a second polypeptide that provides a positive signal to the cell, for example, an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, for example, of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of these (for example, at least a portion of the extracellular domain of any of these), and a second polypeptide that is an intracellular signaling domain described herein (for example, comprising a co-stimulatory domain (for example, 41BB, CD27 or CD28, for example, as described herein) and / or a primary signaling domain (for example, a CD3 zeta signaling domain described herein)).In one embodiment, the agent comprises a first PD1 polypeptide, or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein).

[0032] In another aspect, the invention relates to a method for producing a cell comprising transducing a T cell with a vector comprising a nucleic acid encoding a CAR, for example, a CAR described herein.

[0033] The present invention also provides a method for generating a population of RNA-constructed cells, for example, cells described herein, for example, T cells, transiently expressing exogenous RNA. The method comprises introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises a nucleic acid encoding a CAR molecule described herein. Petition 870260065699, dated 03 / 07 / 2026, page 33 / 532 26 / 250

[0034] In another aspect, the invention relates to a method for conferring antitumor immunity to a mammal, comprising administering to the mammal an effective amount of a cell comprising a CAR molecule, for example, a cell expressing a CAR molecule described herein. In one embodiment, the cell is an autologous T cell. In one embodiment, the cell is an allogeneic T cell. In one embodiment, the mammal is a human being.

[0035] In another aspect, the invention relates to a method for treating a mammal bearing a disease associated with CD19 expression, comprising administering to the mammal an effective amount of the cell comprising a CAR molecule, for example, a CAR molecule described herein.

[0036] In one embodiment, the disease associated with CD19 expression is selected from a proliferative disease, such as cancer or malignancy, or a precancerous condition, such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia, or is a non-cancer-related indication associated with CD19 expression. In one embodiment, the disease is a hematologic cancer. In one embodiment, the hematologic cancer is leukemia.In one modality, the cancer is selected from the group consisting of one or more acute leukemias including, but not limited to, acute lymphoblastic leukemia of B cells (B-ALL), acute lymphoblastic leukemia of T cells (T-ALL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including, but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); hematologic cancers or additional hematologic conditions including, but not limited to, prolymphocytic B-cell leukemia, plasmacytoid dendritic cell blast neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, follicular small cell or large cell lymphoma, lymphoproliferative conditions. Petition 870260065699, dated 03 / 07 / 2026, page 34 / 532 27 / 250 malignant lymphomas, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia and preleukemia, which represents a diverse collection of hematological conditions united by the ineffective production (or dysplasia) of myeloid blood cells, and diseases associated with CD19 expression include, among others, atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative diseases with CD19 expression; and any combination thereof.

[0037] In one embodiment, lymphocyte infusion, for example, allogeneic lymphocyte infusion, is used in cancer treatment, wherein the lymphocyte infusion comprises at least one cell expressing CAR CD19. In another embodiment, autologous lymphocyte infusion is used in cancer treatment, wherein the autologous lymphocyte infusion comprises at least one cell expressing CD19.

[0038] In one embodiment, a cell expressing CAR CD19, for example, a T cell, is administered to an individual who has received a previous stem cell transplant, for example, an autologous stem cell transplant.

[0039] In one embodiment, a cell expressing CAR CD19, for example, a T cell, is administered to an individual who has received a prior dose of melphalan.

[0040] In one embodiment, cells expressing a CAR molecule, for example, a CAR molecule described herein, are administered in combination with an agent that increases the effectiveness of a cell expressing a CAR molecule, for example, an agent described herein.

[0041] In one embodiment, cells expressing a Petition 870260065699, dated 03 / 07 / 2026, p. 35 / 532 28 / 250 molecules of CAR, for example, a CAR molecule described herein, are administered in combination with an agent that improves one or more side effects associated with the administration of a cell expressing a CAR molecule, for example, an agent described herein.

[0042] In one embodiment, cells expressing a CAR molecule, for example, a CAR molecule described herein, are administered in combination with an agent that treats the CD19-associated disease, for example, an agent described herein.

[0043] In one embodiment, cells expressing a CAR molecule, for example, a CAR molecule described herein, are administered at a dose and / or in an administration schedule described herein.

[0044] In one embodiment, the CAR molecule is introduced into T cells, for example, using in vitro transcription, and the individual (e.g., human) receives an initial administration of cells comprising a CAR molecule and one or more subsequent administrations of cells comprising a CAR molecule, wherein one or more subsequent administrations are given less than 15 days apart, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In another embodiment, more than one administration of cells comprising a CAR molecule is given to the individual (e.g., human) per week, for example, 2, 3, or 4 administrations of cells comprising a CAR molecule are given per week.In one embodiment, the individual (e.g., human individual) receives more than one administration of cells comprising a CAR molecule per week (e.g., 2, 3, or 4 administrations per week) (also referred to in this descriptive report as a cycle), followed by one week without administration of cells comprising a molecule. Petition 870260065699, dated 03 / 07 / 2026, page 36 / 532 29 / 250 of CAR and then one or more additional administrations of cells comprising a CAR molecule (e.g., more than one administration of cells comprising a CAR molecule per week) is given to the individual. In another embodiment, the individual (e.g., human individual) receives more than one cycle of cells comprising a CAR molecule, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, cells comprising a CAR molecule are administered every two days, totaling 3 administrations per week. In one embodiment, cells comprising a CAR molecule are administered for at least two, three, four, five, six, seven, eight, or more weeks.

[0045] In one embodiment, cells expressing a CAR molecule, for example, a CAR molecule described herein, are administered as first-line treatment for a disease, for example, cancer, for example, the cancer described herein.In another modality, cells expressing a CAR molecule, for example, a CAR molecule described here, are administered as second-, third-, or fourth-line treatment for a disease, for example, cancer, for example, the cancer described here.

[0046] In one embodiment, a population of cells described herein is managed.

[0047] In another aspect, the invention relates to the isolated nucleic acid molecule encoding a CAR of the invention, to the polypeptide molecule isolated from a CAR of the invention, to the vector comprising a CAR of the invention and to the cell comprising a CAR of the invention for use as a medicament.

[0048] In another aspect, the invention relates to the isolated nucleic acid molecule encoding a CAR of the invention, to the polypeptide molecule isolated from a CAR of the invention, to the vector comprising a CAR of the invention and to the cell comprising a Petition 870260065699, dated 03 / 07 / 2026, page 37 / 532 30 / 250 CAR of the invention for use in the treatment of a disease that expresses CD19.

[0049] In one aspect, the invention includes a population of autologous cells that are transfected or transduced with a vector comprising a nucleic acid molecule encoding a CD19 CAR molecule, for example, as described herein. In one embodiment, the vector is a retroviral vector. In another embodiment, the vector is a self-inactivating lentiviral vector, as described elsewhere in this descriptive report. In one embodiment, the vector is released (e.g., by transfection or electroporation) into a cell, for example, a T cell, wherein the vector comprises a nucleic acid molecule encoding a CD19 CAR molecule as described herein, which is transcribed as an mRNA molecule, and the CD19 CAR molecule is translated from the RNA molecule and expressed on the cell surface.

[0050] In another aspect, the present invention provides a population of cells expressing CARs, for example, CART cells. In some embodiments, the population of cells expressing CARs comprises a mixture of cells expressing different CARs. For example, in one embodiment, the CART cell population may include a first cell expressing a CAR with an anti-CD19 binding domain described herein, and a second cell expressing a CAR with a different anti-CD19 binding domain, for example, an anti-CD19 binding domain described herein that differs from the anti-CD19 binding domain in the CAR expressed by the first cell. As another example, the population of cells expressing CARs may include a first cell expressing a CAR that includes an anti-CD19 binding domain, for example, as described herein, and a second cell expressing a CAR that includes an antigen-binding domain for a target other than CD19 (for example, CD123). In one embodiment, Petition 870260065699, dated 03 / 07 / 2026, page 38 / 532 31 / 250 the population of cells that CAR includes, for example, a first cell that expresses a CAR including a primary intracellular signaling domain, and a second cell that expresses a CAR that includes a secondary signaling domain.

[0051] In another aspect, the present invention provides a population of cells, wherein at least one cell in the population expresses a CAR having an anti-CD19 domain described herein and a second cell expressing another agent, for example, an agent that enhances the activity of a cell expressing a CAR. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, for example, an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, for example, an intracellular signaling domain described herein.In one embodiment, the agent comprises a first polypeptide, for example, of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or a fragment of any of these (for example, at least a portion of the extracellular domain of any of these), and a second polypeptide that is an intracellular signaling domain described herein (for example, comprising a co-stimulatory domain (for example, 41BB, CD27, or CD28, for example, as described herein) and / or a primary signaling domain (for example, a CD3 zeta signaling domain described herein)). In another embodiment, the agent comprises a first polypeptide of PD1, or a fragment thereof (for example, at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (for example, a domain of...). Petition 870260065699, dated 03 / 07 / 2026, page 39 / 532 32 / 250 CD28 signaling described herein and / or a CD3 zeta signaling domain described herein).

[0052] In one embodiment, the nucleic acid molecule encoding a CD19 CAR molecule, for example, as described herein, is expressed as an mRNA molecule. In another embodiment, cells genetically modified to express CD19 CAR, for example, T cells, can be generated by transfection or electroporation of an RNA molecule encoding the desired CARs (e.g., without a vector sequence) into the cell. In another embodiment, a CD19 CAR molecule is translated from the RNA molecule once it is incorporated and expressed on the surface of the recombinant cell. Brief Description of the Drawings

[0053] Figures 1A, 1B and 1C are graphical representations of cytotoxicity, as evaluated in ND317 T cells (normal donor) transduced with mouse anti-CD19 CARs or the humanized anti-CD19 CARs of the invention and cultured with CD19-non-expressing control K562 cells (K562cc), as shown in Figure 1A, CD19-transformed K562 cells (K562.CD19), as shown in Figure 1B, or with malignant B cells isolated from a CLL patient (Pt 14 B cell isolate) as shown in Figure 1C.

[0054] Figures 2A and 2B are graphs showing the proliferative response of humanized and mouse CAR-anti-CD19 expressing cells to CD19+ cells, where the higher number of viable CAR+ T cells correlates with populations showing maximum proliferation of CD4+ and CD8+ cells to primary CLL cells.

[0055] Figure 3 is a graphical representation of the deconvolved HPLC mass spectra for scFvs of the invention, where the upper line depicts the untreated scFv and the lower line depicts the deglycosylated cognate scFv. Petition 870260065699, dated 03 / 07 / 2026, page 40 / 532 33 / 250

[0056] Figure 4 is a graphical representation of conformational stability as measured by Differential Scanning Fluorimetry. The Tm of mouse scFv was 57°C (thick line). All humanized variants of scFv showed higher Tm, around 70°C, when compared to the original mouse scFv. The residues introduced by humanization improved the Tm by more than 10°C.

[0057] Figure 5 is a graphical representation of the proliferation of CAR CD19 transduced T cells, whereby CAR19 cells target (a) a chronic myelogenous leukemia (CML) cell line that is negative for CD19 expression and consequently used as a negative control; (b) recombinant K562 cells positive for CD19 expression and consequently used as a positive control; or (c) Pt14 B cells isolated from a CLL patient that express CD19 on their cell surface.

[0058] Figures 6A and 6B are schematic drawings of representative CARs.

[0059] Figure 7 depicts the progression of cell disease HALLX5447 primary ALL cells in NSG mice after treatment with CD19-specific CAR T cells. The growth of primary human ALL cells in NSG mice after treatment with CD19-specific CAR T cells demonstrated control over disease progression. The mean percentage of CD19+ human ALL cells was an indicator of disease burden in the peripheral blood of NSG mice up to Day 65 after tumor implantation. Black circles: mice treated with 100 μL of PBS via tail vein; red squares: mice treated with sham transduced T cells; blue triangles: mice treated with murine CD19-specific CAR T cells; and inverted purple triangles: mice treated with CAR T cells. Petition 870260065699, dated 03 / 07 / 2026, page 41 / 532 34 / 250 Humanized CD19. Significance calculated by ANOVA; * indicates P<0.01.

[0060] Figure 8 depicts the expression of CD19 in tumor cells from a patient. CD138+ CD45dim tumor cells were stained for CD19 (s-axis) and CD38 (y-axis). Detailed description of the invention Definitions

[0061] Unless otherwise indicated, all technical and scientific terms in this descriptive report have the same meaning as commonly understood by a person skilled in the art to which the invention pertains.

[0062] The term "a" and "an" refers to one or more of (i.e., at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0063] The term "approximately," when referring to a measurable value, such as a quantity, a time duration, and the like, is intended to cover variations of ±20% or, in some cases, ±10% or, in some cases, ±5% or, in some cases, ±1% or, in some cases, ±0.1% of the specified value, as such variations are appropriate for the execution of the disclosed methods.

[0064] The term Chimeric Antigen Receptor or, alternatively, CAR, refers to a recombination-constructed polypeptide comprising at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to in this descriptive report as an intracellular signaling domain) comprising a functional signaling domain derived from a stimulatory molecule as defined below. In one aspect, the stimulatory molecule is the zeta chain associated with the receptor-T cell complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from the Petition 870260065699, dated 03 / 07 / 2026, page 42 / 532 35 / 250 minus a stimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is chosen from 4-1BB (i.e., CD137), CD27, and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule.In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In another aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In another aspect, the CAR comprises an optional leader sequence at the amino (N-ter) termination of the CAR fusion protein.In one aspect, the CAR further comprises a leader sequence at the N-terminal end of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain. Petition 870260065699, dated 03 / 07 / 2026, page 43 / 532 36 / 250 (e.g., aa scFv) during cellular processing and CAR localization to the cell membrane.

[0065] The term signaling domain refers to the functional portion of a protein that acts by transmitting information within the cell to regulate cellular activity through defined signaling pathways by generating second messengers or functioning as an effector in response to such messengers.

[0066] In this descriptive report, the term CD19 refers to the Group of Differentiation protein 19, which is an antigenic determinant detectable in leukemia precursor cells. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found in UniProt / Swiss-Prot under Accession Number P15391, and the nucleotide sequence encoding human CD19 can be found under Accession Number NM_001178098. CD19 is expressed in most B-cell cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Other cells expressing CD19 are provided below in the definition of disease associated with CD19 expression. Furthermore, it is also an early marker of B-cell progenitors. See, for example, Nicholson et al. Mol. Immun.34 (16-17): 1157-1165 (1997). In one aspect, the antigen-binding portion of CART recognizes and binds to an antigen within the extracellular domain of the CD19 protein. In one aspect, the CD19 protein is expressed in a cancer cell.

[0067] The term antibody, in this descriptive report, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that binds specifically to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain. Petition 870260065699, dated 03 / 07 / 2026, p. 44 / 532 37 / 250 or single, or intact immunoglobulins, and may be derived from natural or recombinant sources. Antibodies may be tetramers of immunoglobulin molecules.

[0068] The term antibody fragment refers to at least a portion of an intact antibody, or its recombinant variants, and refers to the antigen-binding domain, for example, an antigenic variable region of an intact antibody that is sufficient to confer specific recognition and binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.The term scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the variable regions of the light and heavy chains are contiguously linked via a short, flexible linker polypeptide and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified in this descriptive report, an scFv may have the variable VL and VH regions in any order, for example, with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or it may comprise VH-linker-VL.

[0069] The CAR composition portion of the invention, comprising an antibody or antibody fragment thereof, may exist in a variety of forms, wherein the antigen-binding domain is expressed as part of a polypeptide chain. Petition 870260065699, dated 03 / 07 / 2026, p. 45 / 532 38 / 250 contiguous, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. In one aspect, the antigen-binding domain of a CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment comprising an scFv.

[0070] The term antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their natural conformations, and which normally determines the class to which the antibody belongs.

[0071] The term antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their natural conformations. Kappa (κ) and lambda (λ) light chains refer to the two main types of antibody light chains.

[0072] The term recombinant antibody refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be interpreted to mean an antibody that has been generated by the synthesis of a DNA molecule encoding the antibody and whose DNA molecule expresses an antibody, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence was obtained using recombinant DNA or amino acid sequence technology that is available and well known in Petition 870260065699, dated 03 / 07 / 2026, p. 46 / 532 39 / 250 technique.

[0073] The term antigen or Ag refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immunologically competent cells, or both. A person skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A person skilled in the art will understand that any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an antigen as that term is used in this descriptive report. Additionally, a person skilled in the art will understand that an antigen does not need to be encoded exclusively by a complete nucleotide sequence of a gene.It is readily apparent that the present invention includes, among other things, the use of partial nucleotide sequences from more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Furthermore, those skilled in the art will understand that an antigen does not need to be encoded by a gene at all. It is readily apparent that an antigen can be generated by synthesis or derived from a biological sample, or it could be a macromolecule other than a polypeptide. Such a biological sample may include, among other things, a tissue sample, a tumor sample, a cell, or a fluid with other biological components.

[0074] The term antitumor effect refers to a biological effect that can manifest itself in various ways, including, among others, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in Petition 870260065699, dated 03 / 07 / 2026, page 47 / 532 40 / 250 life expectancy, decreased proliferation of tumor cells, decreased survival of tumor cells, or improvement of various physiological symptoms associated with the cancerous condition. An antitumor effect may also manifest itself through the ability of the peptides, polynucleotides, cells, and antibodies of the invention to prevent the occurrence of tumors in the first place.

[0075] The term autologous refers to any material derived from the same individual to which it is later reintroduced into the individual.

[0076] The term allogeneic refers to any material derived from an animal other than the same species as the individual to whom the material is reintroduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some respects, allogeneic material from individuals of the same species may be sufficiently distinct genetically to interact antigenically.

[0077] The term xenogeneic refers to a graft derived from an animal of a different species.

[0078] The term cancer refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described here and include, among others, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and similar cancers.

[0079] The term CD19-associated disease includes, among others, a disease associated with CD19 expression or a condition associated with cells expressing CD19, including, for example, proliferative diseases such as cancer or malignancy or Petition 870260065699, dated 03 / 07 / 2026, page 48 / 532 41 / 250 a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia; and a non-cancerous indication associated with cells expressing CD19. In one aspect, a cancer associated with CD19 expression is a hematologic cancer. In one aspect, hematologic cancer is leukemia or lymphoma. In one aspect, a cancer associated with CD19 expression includes cancers and malignancies encompassing, among others, for example, one or more acute leukemias including, among others, for example, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including, among others, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL). Cancers or additional hematological conditions associated with CD19 expression include, among others, for example.B-cell prolymphocytic leukemia, plasmacytoid dendritic cell blast neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia and preleukemia, which represents a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and the like. Other diseases associated with CD19 expression include, among others, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with CD19 expression.Non-cancer indications associated with CD19 expression include, among others, autoimmune diseases (e.g., lupus). Petition 870260065699, dated 03 / 07 / 2026, page 49 / 532 42 / 250 inflammatory disorders (allergies and asthma) and transplants.

[0080] The term conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced by an amino acid residue with a similar side chain. In the art, families of amino acid residues with similar side chains have been identified.These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the invention can be replaced by other amino acid residues from the same side chain family, and the altered CAR can be tested using the functional assays described herein.

[0081] The term stimulation refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, among others, transduction of Petition 870260065699, dated 03 / 07 / 2026, p. 50 / 532 43 / 250 signal via the TCR / CD3 complex. Altered expression of certain molecules, such as the downregulation of TGF-β and / or the reorganization of cytoskeletal structures, and similar factors, can be mediated by stimulation.

[0082] The term stimulatory molecule refers to a molecule expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate(s) the primary activation of the TCR complex in a stimulatory manner for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is inhibited, for example, by the binding of a TCR / CD3 complex to a peptide-laden MHC molecule, leading to the mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a primary signaling domain) that acts in a stimulatory manner may contain a signaling motif known as a tyrosine-based immunoreceptor activating motif or ITAM.Examples of an ITAM containing a primary cytoplasmic signaling sequence of particular use in the invention include, among others, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as ICOS), and CD66d. In a specific CAR of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, for example, a primary CD3-zeta signaling sequence. In a specific CAR of the invention, the primary CD3-zeta signaling sequence is the sequence provided as SEQ ID NO:17, or the equivalent residues of a non-human species, for example, mouse, rodent, monkey, gorilla, and the like. In a specific CAR of the invention, the primary CD3-zeta signaling sequence is the sequence as... Petition 870260065699, dated 03 / 07 / 2026, p. 51 / 532 44 / 250 provided in SEQ ID NO:43, or the equivalent residues of a non-human species, for example, mouse, rodent, monkey, gorilla and the like.

[0083] The term antigen-presenting cell or APC refers to an immune system cell such as an accessory cell (e.g., a B cell, a dendritic cell, and the like) that displays an exogenous antigen complexed with major histocompatibility complexes (MHCs) on its surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process the antigens and present them to T cells.

[0084] An intracellular signaling domain, as the term is used in this descriptive report, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an effector immune function of the CAR-containing cell, for example, a CART cell. Examples of effector immune function, for example, in a CART cell, include cytolytic activity and helper activity, including cytokine secretion.

[0085] In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In another embodiment, the intracellular signaling domain may comprise a co-stimulatory intracellular signaling domain. Exemplary co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory signals or antigen-dependent stimulation. For example, in the case of a CART, a primary intracellular signaling domain may comprise a cytoplasmic sequence of a T-cell receptor, and a co-stimulatory intracellular signaling domain may comprise the Petition 870260065699, dated 03 / 07 / 2026, page 52 / 532 45 / 250 cytoplasmic sequence of the coreceptor or costimulatory molecule.

[0086] A primary intracellular signaling domain may comprise a signaling motif known as a tyrosine-based immunoreceptor activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAM include, among others, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d DAP10 and DAP12.

[0087] The term zeta or, alternatively, zeta chain, CD3zeta or TCR-zeta is defined as the protein provided in GenBank under Accession No. BAG36664.1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, gorilla and the like, and a zeta stimulatory domain or, alternatively, a CD3-zeta stimulatory domain or a TCR-zeta stimulatory domain is defined as the amino acid residues of the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one aspect, the cytoplasmic zeta domain comprises residues 52 to 164 of GenBank under Accession No. BAG36664.1, the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, gorilla and the like, which are its functional orthologs. In one aspect, the zeta stimulatory domain or a CD3-zeta stimulatory domain is the sequence provided as SEQ ID NO:17.In one aspect, the zeta stimulatory domain or a CD3-zeta stimulatory domain is the sequence provided as SEQ ID NO:43.

[0088] The term costimulatory molecule refers to the cognate binding partner in a T molecule that binds specifically with a costimulatory ligand, thereby mediating a molecule response. Petition 870260065699, dated 03 / 07 / 2026, p. 53 / 532 46 / 250 co-stimulatory by T cells, such as, among others, proliferation. Co-stimulatory molecules are molecules on the cell surface, different from antigen receptors or their ligands, that are necessary for an efficient immune response. Co-stimulatory molecules include, among others, an MHC class I molecule, BTLA, and a Toll ligand receptor, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1 BB (CD137).

[0089] An intracellular signaling costimulatory domain may be the intracellular portion of a costimulatory molecule. A costimulatory molecule may be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte signaling activator molecules (SLAM proteins), and NK cell activator receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds to CD83, and the like.

[0090] The intracellular signaling domain may comprise the entire intracellular portion or the entire intracellular signaling domain of the molecule from which it is derived, or a functional fragment thereof.

[0091] The term 4-1BB refers to a member of the superfamily of TNFR with an amino acid sequence provided in GenBank under Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, gorilla, and the like; and a 4-1BB costimulatory domain is defined as amino acid residues 214-255 from GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, gorilla, and the like. In one aspect, the 4-1BB costimulatory domain is the Petition 870260065699, dated 03 / 07 / 2026, p. 54 / 532 47 / 250 sequence provided as SEQ ID NO:16 or the equivalent residues of a non-human species, for example, mouse, rodent, monkey, gorilla and the like.

[0092] The term coding refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates in the synthesis of other polymers and macromolecules in biological processes, having a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the resulting biological properties. Thus, a gene, cDNA, or RNA encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for the transcription of a gene or cDNA, can be referred to as coding for the protein or other product of that gene or cDNA.

[0093] Unless otherwise specified, a nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The expression nucleotide sequence encoding a protein or RNA may also include introns insofar as the protein-coding nucleotide sequence may, in some version, contain one or more introns.

[0094] The term effective amount or therapeutically effective amount are used interchangeably in this descriptive report, and refer to an amount of a compound, formulation, material or composition, as described herein, as effective in achieving Petition 870260065699, dated 03 / 07 / 2026, p. 55 / 532 48 / 250 a specific biological result.

[0095] The term endogenous refers to any material originating from or produced within an organism, cell, tissue, or system.

[0096] The term exogenous refers to any material introduced or produced outside of an organism, cell, tissue, or system.

[0097] The term expression refers to the transcription and / or translation of a given nucleotide sequence directed by a promoter.

[0098] The term transfer vector refers to a composition of matter comprising an isolated nucleic acid that can be used to deliver the nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term transfer vector includes a plasmid or a virus capable of replication. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0099] The term expression vector refers to a vector comprising a recombinant polynucleotide containing expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient active elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all Petition 870260065699, dated 03 / 07 / 2026, page 56 / 532 49 / 250 those known in the art, including cosmids, plasmids (e.g., pure (naked) liposomes or those contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) incorporating recombinant polynucleotides.

[00100] The term lentivirus refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they are able to infect non-dividing cells; they can release a significant amount of genetic information into the host cell's DNA, making them one of the most efficient vector methods for gene delivery. HIV, SIV, and FIV are all examples of lentiviruses.

[00101] The term lentiviral vector refers to a vector derived from at least part of the genome of a lentivirus, including, especially, a self-inactivating lentiviral vector as described in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, among others, for example, the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and similar technologies. Non-clinical types of lentiviral vectors are also available and would be known to those skilled in the art.

[00102] The term homologous or identical refers to the identity of sequence subunits between two polymeric molecules, for example, between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position is occupied in both molecules by the same monomeric subunit; for example, if a position in each of two DNA molecules is occupied by adenine, then these are homologous or identical at that position. Homology between two sequences is a direct function of the number of concordant or homologous positions; for example, if half (by Petition 870260065699, dated 03 / 07 / 2026, p. 57 / 532 50 / 250 example, five positions in a ten-subunit polymer in length) of the positions in two homologous sequences, the two sequences are 50% homologous; if 90% of the positions (for example, 9 out of 10) are concordant or homologous, the two sequences are 90% homologous.

[00103] Humanized forms of non-human antibodies (e.g., murine) are chimeric immunoglobulins, immunoglobulin chains, or their fragments (such as Fv, Fab, Fab', F(ab')2, or other antibody antigen-binding subsequences) that contain the minimal sequence derived from the non-human immunoglobulin. For most, humanized antibodies and their antibody fragments are human immunoglobulins (receptor antibody or antibody fragment) in which residues of a complementarity-determining region (CDR) of the receptor are replaced by residues of a CDR from a non-human species (donor antibody) such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capacity. In some cases, residues of the framework region (FR) of Fv of the human immunoglobulin are replaced by corresponding non-human residues.Furthermore, a humanized antibody / antibody fragment may comprise residues not found in the recipient antibody or in imported CDR or framework sequences. These modifications may further refine and enhance the performance of the antibody or antibody fragment. In general, the humanized antibody or its fragment will comprise substantially all of at least one and typically two variable domains, in which all or substantially all CDR regions correspond to those of a non-human immunoglobulin, and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also... Petition 870260065699, dated 03 / 07 / 2026, page 58 / 532 51 / 250 comprise at least part of a constant region (Fc) of an immunoglobulin, typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[00104] Entirely human refers to an immunoglobulin, such as an antibody or antibody fragment, wherein the molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[00105] The term isolated means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living animal is not isolated, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is isolated. An isolated nucleic acid or protein may exist in substantially pure form, or it may exist in a non-native medium, such as, for example, a host cell.

[00106] In the context of the present invention, the following abbreviations are used for commonly occurring nucleic acid bases. A refers to adenosine, C refers to cytosine, G refers to guanosine, T refers to thymidine, and U refers to uridine.

[00107] The term operationally linked or transcriptional control refers to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the latter. For example, a first nucleic acid sequence is operationally linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operationally linked to a coding sequence if the promoter affects transcription or Petition 870260065699, dated 03 / 07 / 2026, p. 59 / 532 52 / 250 expression of the coding sequence. Operationally linked DNA sequences can be contiguous to each other and, for example, when necessary to join two protein-coding regions, are in the same reading frame.

[00108] The term parenteral administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal, intratumoral or infusion injection techniques.

[00109] The term nucleic acid or polynucleotide refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and their single- or double-stranded polymers. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, a given nucleic acid sequence also implicitly includes its variants resulting from conservative modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence.Specifically, degenerate codon substitutions can be performed by generating sequences in which the third position of one or more selected codons (or all) is replaced by residues with mixed bases and / or deoxynosine (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol Cell.

[00110] The terms peptide, polypeptide, and protein are used interchangeably and refer to a compound formed by amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two Petition 870260065699, dated 03 / 07 / 2026, p. 60 / 532 53 / 250 amino acids, and there is no limitation imposed on the maximum number of amino acids that may comprise the sequence of a protein or peptide. Polypeptides include any protein or peptide comprising amino acids linked together by peptide bonds. In this descriptive report, the term refers to short chains, which are also commonly called peptides, oligopeptides, and oligomers, for example, and to longer chains, which are generally referred to in the art as proteins, of which there are many types. Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[00111] The term promoter refers to a DNA sequence recognized by the cell's synthetic system, or introduced by the synthetic system, necessary to initiate the specific transcription of a polynucleotide sequence.

[00112] The term promoter / regulatory sequence refers to a nucleic acid sequence that is necessary for the expression of a gene product operationally linked to the promoter / regulatory sequence. In some cases, this sequence may be the central promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements that are necessary for the expression of the gene product. The promoter / regulatory sequence may, for example, be the one that expresses the gene product in a tissue-specific manner.

[00113] The term constitutive promoter refers to a nucleotide sequence that, when operationally linked with a polynucleotide encoding or specifying a gene product, causes Petition 870260065699, dated 03 / 07 / 2026, page 61 / 532 54 / 250 that the gene product is produced in a cell under most or all of the cell's physiological conditions.

[00114] The term inducible promoter refers to a nucleotide sequence that, when operationally linked with a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer that matches the promoter is present in the cell.

[00115] The term tissue-specific promoter refers to a nucleotide sequence that, when operationally linked with a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[00116] The term flexible linking polypeptide or linker, when used in the context of an scFv, refers to a linking peptide consisting of amino acids such as glycine and / or serine residues, used alone or in combination to link variable regions of the heavy and light chains together. In one embodiment, the flexible linking polypeptide is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, n=10 (SEQ ID NO:105). In one embodiment, flexible linking polypeptides include, among others, (Gly4 Ser)4 (SEQ ID NO:106) or (Gly4 Ser)3 (SEQ ID NO:107). In another embodiment, the linkers include multiple repetitions of (Gly2 Ser), (GlySer) or (Gly3 Ser) (SEQ ID NO:108). The scope of the invention also covers the linkers described in WO2012 / 138475, which is incorporated herein by reference in this patent application.

[00117] In this descriptive report, a 5' cap (hood) (also Petition 870260065699, dated 03 / 07 / 2026, page 62 / 532 The term 55 / 250 (referred to as RNA cap, 7-methylguanosine RNA cap, or m7G RNA cap) indicates a modified guanine nucleotide that has been added to the 5' end of a eukaryotic messenger RNA immediately after the start of transcription. The 5' cap consists of a terminal group that is attached to the first transcribed nucleotide. Its presence is critical for ribosome recognition and protection by RNases. Cap addition is transcription-coupled, occurring simultaneously with transcription, such that each influences the other. Immediately after the start of transcription, the 5' end of the synthesized mRNA is ligated by a cap-synthesizing complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions necessary for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.The capping portion can be modified to modulate mRNA functionality such as its stability or translation efficiency.

[00118] In this descriptive report, in vitro transcribed RNA refers to RNA, preferably mRNA, that has been synthesized in vitro. In general, in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.

[00119] In this descriptive report, poly(A) is a series of adenosines linked by polyadenylation to mRNA. In the preferred embodiment of a construct for transient expression, polyA contains between 50 and 5,000 (SEQ ID NO: 109), preferably more than 64, more preferably more than 100, more preferably more than 300 or 400 adenosines. Poly(A) sequences can be chemically or enzymatically modified to modulate mRNA functionality such as localization, stability, or translation efficiency.

[00120] In this descriptive report, polyadenylation refers to Petition 870260065699, dated 03 / 07 / 2026, p. 63 / 532 56 / 250 covalent linkage of a polyadenylate moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to pre-mRNA by the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts containing a specific sequence, the polyadenylation signal. The poly(A) tail and the tail-bound protein help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after DNA transcription into RNA, but it can also occur in the cytoplasm.After transcription is complete, the mRNA chain is cleaved by the action of a complex of endonucleases associated with RNA polymerase. The cleavage site is usually characterized by the presence of an AAUAAA base sequence near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.

[00121] In this descriptive report, transient refers to the expression of a non-integrated transgene for a period of hours, days, or weeks, where the time period of expression is shorter than the time period for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.

[00122] The term signal transduction pathway refers to the biochemical relationship between a variety of signal transducing molecules that participate in the transmission of a signal from one part of a cell to another part of a cell. The expression cell surface receptor includes molecules and complexes of molecules capable of receiving Petition 870260065699, dated 03 / 07 / 2026, page 64 / 532 57 / 250 a signal and to transmit the signal across a cell membrane.

[00123] The term individual is intended to include living organisms in which an immune response can be elicited (e.g., mammals, humans).

[00124] The term, a substantially purified cell, refers to a cell that is essentially free of other cell types. A substantially purified cell may also refer to a cell that has been separated from other cell types with which it is normally associated in its natural state. In some cases, a population of substantially purified cells refers to a homogeneous cell population. In other cases, this term simply refers to a cell that has been separated from the cells with which it is naturally associated in its natural state. In some respects, the cells are cultured in vitro. In other respects, the cells are not cultured in vitro.

[00125] The term therapeutic, in this descriptive report, means a treatment. A therapeutic effect is obtained by reducing, suppressing, remitting, or eradicating a disease state.

[00126] The term prophylaxis, in this descriptive report, means the prevention or preventive treatment for a disease or disease state.

[00127] In the context of the present invention, tumor antigen or hyperproliferative disorder antigen or antigen associated with a hyperproliferative disorder refers to antigens that are common to specific hyperproliferative disorders. In certain respects, the hyperproliferative disorder antigens of the present invention are derived from cancers, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and Petition 870260065699, dated 03 / 07 / 2026, page 65 / 532 58 / 250 adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer and similar cancers.

[00128] The term transfected or transformed or transduced refers to a process by which an exogenous nucleic acid is transferred or introduced into the host cell. A transfected or transformed or transduced cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary cell in question and its progeny.

[00129] The term ligand specifically refers to an antibody, or ligand, that recognizes and binds to the cognate binding protein partner (e.g., a stimulatory molecule and / or co-stimulatory molecule present on a T cell) present in a sample, but whose antibody or ligand does not substantially recognize or bind to other molecules in the sample.

[00130] Ranges: Throughout this descriptive report, various aspects of the invention may be presented in range format. It should be understood that the description in range format is simply for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of the invention. Thus, a described range is considered to specifically encompass all possible sub-ranges, as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to specifically reveal sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1; 2; 2.7; 3; 5; 5; 5, 3 and 6. As another example, a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity.This applies regardless of the range width. Description. Petition 870260065699, dated 03 / 07 / 2026, page 66 / 532 59 / 250

[00131] The present invention provides material compositions and methods of use for the treatment of a disease, such as cancer, using humanized anti-CD19 antigen chemical receptors (CARs).

[00132] In one aspect, the invention provides a series of chimeric antigen receptors (CARs) comprising an antibody or antibody fragment constructed to enhance binding to a CD19 protein. In one aspect, the invention provides a cell (e.g., T cell) constructed to express a CAR, wherein the CAR T cell (CART) exhibits an antitumor property. In one aspect, a cell is transformed with the CAR and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., T cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express the CAR. In another embodiment, the cell (e.g., T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR. In some such embodiments, the cell can transiently express the CAR.

[00133] In one aspect, the anti-CD19 protein-binding portion of CAR is an scFv antibody fragment. In one aspect, such antibody fragments are functional in that they retain equivalent binding affinity, i.e., they bind to the same antigen with comparable efficacy as the IgG antibody from which they are derived. In one aspect, such antibody fragments are functional in that they provide a biological response that may include, among others, activation of an immune response, inhibition of signal transduction originating from their antigenic target, inhibition of kinase activity, and the like, as will be understood by Petition 870260065699, dated 03 / 07 / 2026, page 67 / 532 60 / 250 technical subject matter expert. In one aspect, the anti-CD19 antigen-binding domain of CAR is an scFv antibody fragment that is humanized when compared to the murine scFv sequence from which it is derived. In one aspect, the original murine scFv sequence is the CAR19 construct provided in PCT Publication WO2012 / 079000 and provided here as SEQ ID NO:58.

[00134] In some aspects, the antibodies of the invention are incorporated into a chimeric antigen receptor (CAR). In one aspect, the CAR comprises the polypeptide sequence provided as SEQ ID NO: 12 in PCT Publication WO2012 / 079000, and provided herein as SEQ ID NO: 58, wherein the scFv domain is replaced by one or more sequences selected from SEQ ID NOS: 1-12. In one aspect, the scFv domains of SEQ ID NOS: 1-12 are humanized variants of the scFv domain of SEQ ID NO: 59, which is a murine-origin scFv fragment that specifically binds to human CD19. Humanization of this mouse scFv may be desirable for the clinical setting, where mouse-specific residues could induce a human anti-mouse antigen (HAMA) response in patients receiving CART19 treatment, for example, treatment with T cells transduced with the CAR19 construct.

[00135] In one aspect, the anti-CD19 binding domain, for example, the humanized scFv portion of a CAR of the invention, is encoded by a transgene whose sequence codons have been optimized for expression in a mammalian cell. In another aspect, the entire CAR construct of the invention is encoded by a transgene whose entire sequence codons have been optimized for expression in a mammalian cell. Codon optimization refers to the finding that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is influenced by different species. Such Petition 870260065699, dated 03 / 07 / 2026, page 68 / 532 61 / 250 codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods are known in the art, and include, for example, the methods described in at least U.S. Patents Numbers 5,786,464 and 6,114,148.

[00136] In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:1. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:2. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:3. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:4. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:5. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:6. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:7. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:8. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:9. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 10. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 11.In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:12.

[00137] In one aspect, the CARs of the invention combine an antigen-binding domain of a specific antibody with an intracellular signaling molecule. For example, in some aspects, the intracellular signaling molecule includes, among others, the CD3-zeta, 4-1BB, and CD28 chain signaling modules and combinations thereof. In one aspect, the antigen-binding domain binds to CD19. In one aspect, the CD19 CAR comprises a CAR selected from the sequence provided in one or more of the SEQs. Petition 870260065699, dated 03 / 07 / 2026, page 69 / 532 62 / 250 ID NOS: 31 - 42. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:31. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:32. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:33. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:34. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:35. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:36. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:37. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:38. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:39. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:40. In one aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:41. In another aspect, CAR CD19 comprises the sequence provided in SEQ ID NO:42.

[00138] Furthermore, the present invention provides CAR CD19 compositions and their use in medicines or methods for treating, among other things, diseases, cancer or any malignancy or autoimmune diseases involving cells or tissues that express CD19.

[00139] In one aspect, the CAR of the invention can be used to eradicate normal cells expressing CD19, whereby it is applicable for use as cell conditioning therapy prior to cell transplantation. In one aspect, the normal cell expressing CD19 is a normal stem cell expressing CD19 and the cell transplant is a stem cell transplant.

[00140] In one aspect, the invention provides a cell (e.g., T cell) constructed to express a chimeric antigen receptor (CAR), wherein the CAR T cell (CART) exhibits an antitumor property. A preferred antigen is CD19. In one aspect, the antigen-binding domain of the CAR comprises a fragment of Petition 870260065699, dated 03 / 07 / 2026, page 70 / 532 63 / 250 partially humanized anti-CD19 antibody. In one aspect, the antigen-binding domain of the CAR is a partially humanized anti-CD19 antibody fragment including a scFv. Thus, the invention provides a CD19 CAR comprising a humanized anti-CD19 binding domain and constructed on a T cell and methods of its use for adoptive therapy.

[00141] In one aspect, the CAR CD19 comprises at least one intracellular domain selected from the group consisting of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3zeta signaling domain, and any combination thereof. In one aspect, the CAR CD19 comprises at least one intracellular signaling domain that originates from one or more costimulatory molecules, excluding CD137 (4-1BB) or CD28. Chimeric antigen receptor (CAR)

[00142] The present invention encompasses a constructed recombinant DNA comprising coding sequences of a CAR, wherein the CAR comprises a humanized antibody fragment that specifically binds to CD19, for example, human CD19, wherein the antibody fragment sequence is contiguous and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain may comprise a costimulatory molecule signaling domain and / or a primary signaling domain, for example, a zeta chain. The costimulatory molecule signaling domain refers to a portion of the CAR comprising at least one portion of the intracellular domain of a costimulatory molecule.

[00143] In specific aspects, a CAR constructed of the invention comprises an scFv domain selected from the group consisting of SEQ ID NOS:1-12, wherein the scFv may be preceded by an optional leader sequence, as provided in SEQ ID NO: 13, and Petition 870260065699, dated 03 / 07 / 2026, page 71 / 532 64 / 250 followed by an optional articulation sequence as provided in SEQ ID NO:14, SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:49, a transmembrane region as provided in SEQ ID NO:15, an intracellular signaling domain that includes SEQ ID NO:16 or SEQ ID NO:51 and a CD3 zeta sequence that includes SEQ ID NO:17 or SEQ ID NO:43, where the domains are contiguous and in the same reading frame to form a single fusion protein. The invention also includes a nucleotide sequence that encodes the polypeptide of each of the scFv fragments, selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.The invention also includes a nucleotide sequence encoding the polypeptide of each of the scFv fragments, selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, and each of the domains of SEQ ID NOS: 13-17, plus the CAR CD19 fusion protein encoded in the invention. In one aspect, exemplary constructs of CAR CD19 comprise an optional leader sequence, an extracellular antigen-binding domain, a linkage, a transmembrane domain, and an intracellular stimulatory domain. In one aspect, an exemplary CAR CD19 construct comprises an optional leader sequence, an extracellular antigen-binding domain, an articulation, a transmembrane domain, an intracellular costimulatory domain, and an intracellular stimulatory domain.Specific constructs of CAR CD19, containing humanized scFv domains of the invention, are provided as SEQ ID NOS: 31-42.

[00144] Complete CAR sequences are also provided here as SEQ ID NOS: 31-42, as shown in Table 3. Petition 870260065699, dated 03 / 07 / 2026, page 72 / 532 65 / 250

[00145] An exemplary leader sequence is provided as SEQ ID NO: 13. An exemplary link / spacer sequence is provided as SEQ ID NO: 14, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 15. An exemplary sequence of the intracellular signaling domain of protein 4-1BB is provided as SEQ ID NO: 16. An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO: 51. An exemplary sequence of the CD3zeta domain is provided as SEQ ID NO: 17 or SEQ ID NO: 43.

[00146] In one aspect, the present invention encompasses a recombinant nucleic acid construct comprising a CAR-encoding nucleic acid molecule, wherein the nucleic acid molecule comprises the nucleic acid sequence encoding an anti-CD19 binding domain, for example, described herein, which is contiguous and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. In one aspect, the anti-CD19 binding domain is selected from one or more of the SEQ IDs NOS:1-12. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of the sequence provided in one or more of the SEQ IDs NOS:61-72. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of the SEQ ID NO:61. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:62.In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:63. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:64. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:65. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813. Petition 870260065699, dated 03 / 07 / 2026, page 73 / 532 66 / 250 of SEQ ID NO:66. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:67. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:68. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:69. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:70. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:71. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:72.

[00147] In one aspect, the present invention encompasses a recombinant nucleic acid construct comprising a transgene encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an anti-CD19 binding domain, selected from one or more of the SEQ IDs NOS:61-72, wherein the sequence is contiguous and in the same reading frame as the nucleic acid sequence encoding an intracellular signaling domain. An exemplary intracellular signaling domain that may be used in the CAR includes, among others, one or more intracellular signaling domains of, for example, CD3-zeta, CD28, 4-1BB and the like. In some cases, the CAR may comprise any combination of CD3-zeta, CD28, 4-1BB and the like. In one aspect, the nucleic acid sequence of a CAR construct of the invention is selected from one or more of the SEQ IDs NOS:8596.In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:85. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:86. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:87. In one aspect, the nucleic acid sequence of a... Petition 870260065699, dated 03 / 07 / 2026, p. 74 / 532 67 / 250 CAR construct is SEQ ID NO:88. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:89. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:90. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:91. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:92. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:93. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:94. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:95. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:96. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:97. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:98.In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:99.

[00148] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as screening libraries of cells expressing the gene, deriving the gene from a vector known to contain it, or isolating it directly from cells and tissues containing it, using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically instead of being cloned.

[00149] The present invention includes retroviral and lentiviral vector constructs expressing a CAR that can be transduced directly into a cell.

[00150] The present invention also includes an RNA construct that can be transfected directly into a cell. One method for generating mRNA to be used in transfection involves in vitro transcription. Petition 870260065699, dated 03 / 07 / 2026, p. 75 / 532 68 / 250 (IVT) of a template with specially designed primers, followed by the addition of polyA, to produce a construct containing untranslated sequences (UTRs) at 3' and 5', a 5' cap and / or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO:118). The RNA thus produced can be efficiently transfected into different cell types. In one embodiment, the template includes sequences for the CAR. In another embodiment, a CAR RNA vector is transduced into a cell by electroporation. Antigen-binding domain

[00151] In one aspect, the CAR of the invention comprises a specific target binding element, also designated an antigen-binding domain. The choice of portion depends on the type and number of ligands that define the surface of a target cell. For example, the antigen-binding domain may be chosen to recognize a ligand that acts as a marker on the cell surface of target cells associated with a particular disease state. Thus, examples of cell surface markers that act as ligands for the antigen-binding domain in a CAR of the invention include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[00152] In one aspect, the CAR-mediated T cell response can be directed to an antigen of interest by constructing an antigen-binding domain that binds specifically to a desired antigen in the CAR.

[00153] In one aspect, the CAR portion comprising the antigen-binding domain comprises an antigen-binding domain targeting CD19. In one aspect, the antigen-binding domain targets human CD19. In one aspect, the antigen-binding domain Petition 870260065699, dated 03 / 07 / 2026, page 76 / 532 The 69 / 250 CAR antigen has binding specificity equal to or similar to that of the FMC63 scFv fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997).

[00154] The antigen-binding domain may be any domain that binds to the antigen, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody and its functional fragments, including, but not limited to, a single-domain antibody such as a variable heavy chain (VH) domain, a variable light chain (VL) domain and a variable (VHH) domain of a camelid-derived nanobody, and an alternative scaffold known in the art as an antigen-binding domain, such as a recombinant fibronectin domain, and the like. In some cases, it is beneficial that the antigen-binding domain be derived from the same species in which the CAR will ultimately be used. For example, for human use, it may be beneficial that the antigen-binding domain of the CAR comprise human or humanized residues for the antigen-binding domain of an antibody or antibody fragment.

[00155] Thus, in one aspect, the antigen-binding domain comprises a humanized antibody or antibody fragment. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) of the complementarity-determining region 1 of the light chain (LC CDR1), the complementarity-determining region 2 of the light chain (LC CDR2), and the complementarity-determining region 3 of the light chain (LC CDR3) of a humanized anti-CD19 binding domain described herein, and / or one or more (e.g., all three) of the complementarity-determining region 1 of the heavy chain (HC CDR1), the complementarity-determining region 2 of the heavy chain (HC CDR2), and the complementarity-determining region 3 of the light chain Petition 870260065699, dated 03 / 07 / 2026, page 77 / 532 70 / 250 heavy chain (HC CDR3) of a humanized anti-CD19 binding domain described herein, for example, a humanized anti-CD19 binding domain comprising one or more, for example, all three, LC CDRs and one or more, for example, all three, HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (for example, all three) between the complementarity-determining region 1 of the heavy chain (HC CDR1), the complementarity-determining region 2 of the heavy chain (HC CDR2) and the complementarity-determining region 3 of the heavy chain (HC CDR3) of a humanized anti-CD19 binding domain described herein, for example, the humanized anti-CD19 binding domain has two variable regions of the heavy chain, each comprising an HC CDR1, an HC CDR2 and an HC CDR3 described herein.In one embodiment, the humanized anti-CD19 binding domain comprises a variable region of the humanized light chain described herein (e.g., in Table 3) and / or a variable region of the humanized heavy chain described herein (e.g., in Table 3). In one embodiment, the humanized anti-CD19 binding domain comprises a variable region of the humanized heavy chain described herein (e.g., in Table 3), for example, at least two variable regions of the humanized heavy chain described herein (e.g., in Table 3). In one embodiment, the anti-CD19 binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence from Table 3.In one embodiment, the anti-CD19 binding domain (e.g., an scFv) comprises: a variable light chain region comprising an amino acid sequence with at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence from a variable light chain region provided in Table 3, or a sequence. Petition 870260065699, dated 03 / 07 / 2026, page 78 / 532 71 / 250 with 95-99% identity to an amino acid sequence from Table 3; and / or a variable region of the heavy chain comprising an amino acid sequence with at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence from a variable region of the heavy chain provided in Table 3, or a sequence with 95-99% identity with respect to an amino acid sequence from Table 3. In one embodiment, the humanized anti-CD19 binding domain comprises a sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or a sequence with 95-99% identity with these.In one embodiment, the nucleic acid sequence encoding the anti-CD19 humanized binding domain comprises a sequence selected from a group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, or a sequence with 95-99% identity to these. In one embodiment, the anti-CD19 humanized binding domain is an scFv, and a variable region of the light chain comprising an amino acid sequence described herein, for example, in Table 3, is linked to a variable region of the heavy chain comprising an amino acid sequence described herein, for example, in Table 3, via a linker, for example, a linker described herein. In one embodiment, the humanized anti-CD19 linking domain includes a linker (Gly4-Ser)n, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO:53).The variable region of the light chain and the variable region of the heavy chain of a scFv can be, for example, in any of the following orientations: variable region of the light chain-linker-region. Petition 870260065699, dated 03 / 07 / 2026, page 79 / 532 72 / 250 variable of the heavy chain or variable region of the heavy chain-linker / variable region of the light chain.

[00156] In one aspect, the antigen-binding domain portion comprises one or more sequences selected from SEQ ID NOS:1-12. In one aspect, the humanized CAR is selected from one or more sequences selected from SEQ ID NOS:31-42. In some aspects, a non-human antibody is humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human being, or a fragment thereof. In one aspect, the antigen-binding domain is humanized.

[00157] A humanized antibody can be produced using a variety of techniques known in the art, including, among others, CDR grafting (see, for example, European Patent No. EP 239 400; International Publication No. WO 91 / 09967; and US Patents Nos. 5 225 539, 5 530 101 and 5 585 089, the contents of which are incorporated herein in their entirety by reference in this patent application), veneering or resurfacing (see, for example, European Patents Nos. EP 592 106 and EP 519 596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973, the contents of which are incorporated herein in their entirety by reference in this patent application), chain shuffling (see, for example, US PatentNo. 5,565,332, the contents of which are incorporated herein in their entirety by reference in this patent application) and techniques described in, for example, U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng.,. Petition 870260065699, dated 03 / 07 / 2026, p. 80 / 532 73 / 250 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 (Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994) and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), the contents of which are incorporated herein, in their entirety, by reference in this patent application. Frequently, the framework residues in the framework regions will be replaced by the corresponding residue of the CDR donor antibody to alter, for example, improve, the binding to the antigen. These substitutions in the scaffold are identified by methods well known in the art, for example, by modeling the interactions of CDR and scaffold residues to identify framework residues important for antigen binding and by sequence comparison to identify unusual scaffold residues at specific positions (See, for example, Queen et al., US Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, the contents of which are incorporated herein in their entirety by reference in this patent application).

[00158] A humanized antibody or antibody fragment has one or more amino acid residues remaining from a non-human source. These non-human amino acid residues are often referred to as import residues, which are typically taken from a variable import domain. As provided in the invention, humanized antibodies or antibody fragments comprise one or more CDRs of non-human immunoglobulin molecules and framework regions, wherein the amino acid residues comprising the framework are derived wholly or mainly from human germline. Multiple techniques for the humanization of antibodies or antibody fragments are Petition 870260065699, dated 03 / 07 / 2026, page 81 / 532 74 / 250 well known in the field and can be performed essentially following the method of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody, i.e., CDR grafting (EP 239 400; PCT Publication No. WO 91 / 09967; and US Patents Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are here). incorporated, in their entirety, by reference in this patent application). In such humanized antibodies and antibody fragments, substantially less than one intact human variable domain has been replaced by the corresponding sequence from a non-human species.Humanized antibodies are frequently human antibodies in which some CDR residues and possibly some framework (FR) residues are replaced by analogous site residues in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by faceting or remodeling (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or by chain scrambling (US Patent No. 5,565,332), the contents of which are incorporated herein in their entirety by reference in this patent application.

[00159] The choice of human variable domains, both light and heavy chain, to be used in the creation of humanized antibodies aims to reduce antigenicity. According to the so-called best-fit method, the variable domain sequence of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that most closely matches that of the rodent is accepted as the Petition 870260065699, dated 03 / 07 / 2026, page 82 / 532 75 / 250 human scaffold (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein in their entirety by reference in this patent application). Another method makes use of a specific scaffold derived from the consensus sequence in all human antibodies of a given subset of light or heavy chains. The same framework can be used for several different humanized antibodies (see, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein in their entirety by reference in this patent application). In some embodiments, the structure region, for example, all four structure regions, of the variable region of the heavy chain are derived from a VH4_4-59 germline sequence.In one embodiment, the framework region may comprise one, two, three, four, or five modifications, for example, substitutions, for example, of the amino acid in the corresponding murine sequence (e.g., of SEQ ID NO:58). In one embodiment, the framework region, for example, all four framework regions of the variable region of the light chain are derived from a germline sequence VK3_1.25. In one embodiment, the framework region may comprise one, two, three, four, or five modifications, for example, substitutions, for example, of the amino acid in the corresponding murine sequence (e.g., of SEQ ID NO:58).

[00160] In some respects, the portion of a CAR composition of the invention comprising an antibody fragment is humanized with high affinity retention for the antigenic target and other favorable biological properties.According to one aspect of the invention, humanized antibodies and antibody fragments are prepared by a process of analyzing the original sequences and... Petition 870260065699, dated 03 / 07 / 2026, page 83 / 532 76 / 250 several conceptual humanized products, using three-dimensional models of the original and humanized sequences. Three-dimensional models of immunoglobulins are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the probable function of residues in the functioning of the candidate immunoglobulin sequence, for example, analysis of residues that influence the ability of the candidate immunoglobulin to bind to the target antigen. In this way, FR residues can be selected and combined from the receptor sequences and imported so that the desired characteristic of the antibody or antibody fragment, such as increased affinity for the target antigen, is achieved.In general, CDR residues are directly and mostly substantially involved in influencing antigen binding.

[00161] A humanized antibody or antibody fragment may retain antigenic specificity similar to that of the original antibody, for example, in the present invention, the ability to bind to human CD19. In some embodiments, a humanized antibody or antibody fragment may have improved binding affinity and / or specificity for human CD19.

[00162] In one aspect, the anti-CD19 binding domain is characterized by functional attributes or specific properties of an antibody or antibody fragment. For example, in one aspect, the portion of a CAR composition of the invention comprising an antigen-binding domain binds specifically to human CD19. In one aspect, the antigen-binding domain has binding specificity to human CD19 equal to or similar to that of Petition 870260065699, dated 03 / 07 / 2026, page 84 / 532 77 / 250 FMC63 scFv described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the invention relates to an antigen-binding domain comprising an antibody or antibody fragment, wherein the antibody-binding domain specifically binds to a CD19 protein or a fragment thereof, wherein the antibody or antibody fragment comprises a variable light chain and / or a variable heavy chain that includes an amino acid sequence of SEQ ID NO: 1-12. In one aspect, the antigen-binding domain comprises an amino acid sequence of an scFv selected from SEQ ID NOs: 1-12. In certain aspects, the scFv is contiguous and in the same reading frame as a leader sequence. In one aspect, the leader sequence is the polypeptide sequence provided as SEQ ID NO: 13.

[00163] In one aspect, the anti-CD19 binding domain is a fragment, for example, a single-strand variable fragment (scFv). In one aspect, the anti-CD19 binding domain is an Fv, a Fab, a (Fab')2 or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and their fragments of the invention bind to a wild-type or enhanced-type CD19 protein with affinity.

[00164] In some cases, scFvs can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be produced by linking VH and VL regions together using flexible linking polypeptides. The scFv molecules comprise a linker (e.g., a Ser-Gly linker) with optimized length and / or amino acid composition. The length of the linker can greatly affect how the variable regions of an scFv will fold and interact. In fact, if a short linking polypeptide is employed (e.g., Petition 870260065699, dated 03 / 07 / 2026, page 85 / 532 78 / 250 between 5-10 amino acids), internal folding of the chains is prevented. Folding between chains is also necessary to join the two variable regions and form a functional epitope binding site. For examples of linker orientation and size, see, for example, Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, a Publication of US Patent Application Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794 and PCT Publications Nos. WO2006 / 020258 and WO2007 / 024715, the contents of which are incorporated herein by reference in this patent application.

[00165] An scFv may comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In another embodiment, the linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1 (SEQ ID NO:18). In one embodiment, the linker can be (Gly4Ser)4 (SEQ ID NO:106) or (Gly4Ser)3 (SEQ ID NO:107). The variation in the linker length can retain or reinforce the activity, resulting in superior efficacy in activity studies. Stability and mutations

[00166] The stability of an anti-CD19 binding domain, for example, scFv molecules (e.g., soluble scFv) can be evaluated with reference to the biophysical properties (e.g., thermal stability) of a conventional scFV control molecule or a complete antibody. In one embodiment, the thermal stability of the humanized scFv is approximately 0.1, approximately 0.25, approximately 0.5, approximately 0.75, approximately 1, approximately 1.25, approximately 1.5, Petition 870260065699, dated 03 / 07 / 2026, page 86 / 532 79 / 250 approximately 1.75, approximately 2, approximately 2.5, approximately 3, approximately 3.5, approximately 4, approximately 4.5, approximately 5, approximately 5.5, approximately 6, approximately 6.5, approximately 7, approximately 7.5, approximately 8, approximately 8.5, approximately 9, approximately 9.5, approximately 10 degrees, approximately 11 degrees, approximately 12 degrees, approximately 13 degrees, approximately 14 degrees or approximately 15 degrees Celsius greater than that of a control binding molecule (e.g., a conventional scFv molecule) in the assays described.

[00167] The improved thermal stability of the anti-CD19 binding domain, e.g., scFv, is subsequently conferred to the entire CART19 construct, leading to improved therapeutic properties of the CART19 construct. The thermal stability of the anti-CD19 binding domain, e.g., scFv, can be improved by at least approximately 2°C or 3°C when compared to that of a conventional antibody. In one embodiment, the anti-CD19 binding domain, e.g., scFv, exhibits improved thermal stability of 1°C when compared to a conventional antibody. In another embodiment, the anti-CD19 binding domain, e.g., scFv, exhibits improved thermal stability of 2°C when compared to a conventional antibody. In yet another embodiment, scFv exhibits improved thermal stability of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15°C when compared to a conventional antibody.Comparisons can be made, for example, between the scFv molecules described here and scFv molecules or Fab fragments of an antibody from which the VH and VL of scFv were derived. Thermal stability can be assessed using methods known in the art. For example, in one embodiment, Tm values ​​can be measured. Methods for measuring... Petition 870260065699, dated 03 / 07 / 2026, page 87 / 532 80 / 250 Tm and other methods for determining protein stability are described in more detail below.

[00168] Mutations in scFv (resulting from humanization or direct mutagenesis of soluble scFv) alter the stability of scFv and improve the overall stability of scFv and the CART19 construct. The stability of humanized scFv is compared against murine scFv by means of measurements such as Tm, denaturation temperature and aggregation temperature.

[00169] The binding capacity of mutant scFvs can be determined using the assays described in the Examples.

[00170] In one embodiment, the anti-CD19 binding domain, for example, scFv, comprises at least one mutation resulting from the humanization process, such that the mutant scFv confers improved stability to the CART19 construct. In another embodiment, the anti-CD19 binding domain, for example, scFv, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations resulting from the humanization process such that the mutant scFv confers improved stability to the CART19 construct. Methods for evaluating protein stability

[00171] The stability of an antigen-binding domain can be assessed using, for example, the methods described below. Such methods allow the determination of multiple thermal transitions in unfolding, where the least stable domain unfolds first or limits the overall stability threshold of a cooperatively unfolding multidomain unit (e.g., a multidomain protein exhibiting a single unfolding transition). The least stable domain can be identified by some additional methods. Mutagenesis can be performed to probe which domain limits overall stability. Additionally, the protease resistance of a multidomain protein can be Petition 870260065699, dated 03 / 07 / 2026, page 88 / 532 81 / 250 performed under conditions in which it is known, via DSC or other spectroscopic methods, that the less stable domain will unfold intrinsically (Fontana, et al., (1997) Fold. Des., 2: R17-26; Dimasi et al. (2009) J. Mol. Biol. 393: 672-692). Once the less stable domain is identified, the sequence encoding that domain (or part of it) can be used as a test sequence in the methods. a) Thermal stability

[00172] The thermal stability of the compositions can be analyzed using some non-limiting biophysical or biochemical techniques known in the field. In certain embodiments, thermal stability is evaluated by analytical spectroscopy.

[00173] An exemplary method of analytical spectroscopy is Differential Scanning Calorimetry (DSC). DSC employs a calorimeter that is sensitive to the heat absorbances that accompany the unfolding of most proteins or protein domains (see, for example, Sanchez-Ruiz, et al., Biochemistry, 27: 1648-52, 1988). To determine the thermal stability of a protein, a sample of the protein is placed in the calorimeter and the temperature is raised until the Fab or scFv unfolds. The temperature at which the protein unfolds is indicative of the overall stability of the protein.

[00174] Another exemplary method of analytical spectroscopy is Circular Dichroism (CD) spectroscopy. CD spectrometry measures the optical activity of a composition as a function of increasing temperature. Circular dichroism (CD) spectroscopy measures differences arising in the absorption of left-polarized light versus right-polarized light that are due to structural asymmetry. A disorganized or unfolded structure results in a CD spectrum very different from that of an organized or folded structure. The CD spectrum reflects the sensitivity of proteins to the denaturing effects of increasing temperature and is therefore indicative of Petition 870260065699, dated 03 / 07 / 2026, p. 89 / 532 82 / 250 thermal stability of a protein (see, van Mierlo and Steemsma, J. Biotechnol., 79(3):281-98, 2000).

[00175] Another exemplary analytical spectroscopy method for measuring thermal stability is Fluorescence Emission Spectroscopy (see, van Mierlo and Steemsma, supra). Yet another exemplary analytical spectroscopy method for measuring thermal stability is Nuclear Magnetic Resonance (NMR) Spectroscopy (see, for example, van Mierlo and Steemsma, supra).

[00176] The thermal stability of a composition can be measured biochemically. An exemplary biochemical method for assessing thermal stability is a thermal challenge assay. In a thermal challenge assay, a composition is subjected to a range of elevated temperatures for a specified period of time. For example, in one embodiment, test molecules of scFv or molecules comprising scFv molecules are subjected to a range of increasing temperatures, for example, for 1–1.5 hours. The protein activity is then assessed by a relevant biochemical assay. For example, if the protein is a binding protein (e.g., scFv or a polypeptide containing scFv), the binding activity of the binding protein can be determined by a functional or quantitative ELISA assay.

[00177] Such an assay can be performed in high-throughput format and those described in the Examples using E. coli and high-throughput screening. A library of anti-CD19 binding domains, for example, scFv variants, can be created using methods known in the art. The expression of the anti-CD19 binding domain, for example, scFv, can be induced and the anti-CD19 binding domain, for example, scFv, can be subjected to thermal challenge. Samples tested in the challenge can be analyzed for binding, and those anti-CD19 binding domains, for example, scFvs, that are stable can be identified. Petition 870260065699, dated 03 / 07 / 2026, page 90 / 532 83 / 250 can be gradually expanded and characterized in more detail.

[00178] Thermal stability is assessed by measuring the melting temperature (Tm) of a composition using any of the above techniques (e.g., analytical spectroscopy techniques). The melting temperature is the temperature at the midpoint of a thermal transition curve, where 50% of the molecules in a composition are in the folded state (See, for example, Dimasi et al. (2009) J. Mol Biol. 393: 672-692). In one embodiment, the Tm values ​​for an anti-CD19 binding domain, e.g., scFv, are close to 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C. In one modality, the Tm values ​​for an IgG are close to 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C. 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C. In one embodiment, the Tm values ​​for a multivalent antibody are close to 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C.

[00179] Thermal stability is also assessed by measuring heat Petition 870260065699, dated 03 / 07 / 2026, page 91 / 532 84 / 250 specific heat capacity (Cp) of a composition using an analytical calorimetric technique (e.g., DSC). The specific heat of a composition is the energy (e.g., in kcal / mol) required to raise the temperature of 1 mol of water by 1°C. A large Cp value is a hallmark of a denatured composition or inactive protein. The change in heat capacity (ΔCp) of a composition is measured by determining the specific heat of a composition before and after its thermal transition. Thermal stability can also be assessed by measuring or determining other thermodynamic stability parameters, including Gibbs free energy of splitting (ΔG), enthalpy of splitting (ΔH), or entropy of splitting (ΔS). One or more of the above biochemical assays (e.g., a thermal challenge assay) are used to determine the temperature (i.e., the TC value) at which 50% of the composition retains its capacity (e.g., binding activity).

[00180] Furthermore, mutations to the anti-CD19 binding domain, e.g., scFv, alter the thermal stability of the anti-CD19 binding domain, e.g., scFv, when compared to the non-mutant anti-CD19 binding domain, e.g., scFv. When the humanized anti-CD19 binding domain, e.g., scFv, is incorporated into a CART19 construct, the humanized anti-CD19 binding domain, e.g., scFv, confers thermal stability to the overall anti-CD19 CART construct. In one embodiment, the anti-CD19 binding domain, e.g., scFv, comprises a single mutation that confers thermal stability to the anti-CD19 binding domain, e.g., scFv. In another embodiment, the anti-CD19 binding domain, e.g., scFv, comprises multiple mutations that confer thermal stability to the anti-CD19 binding domain, e.g., scFv. In one embodiment, multiple mutations in the anti-CD19 binding domain, for example, scFv, have an additive effect. Petition 870260065699, dated 03 / 07 / 2026, page 92 / 532 85 / 250 the thermal stability of the anti-CD19 binding domain, for example, scFv. b) % of aggregation

[00181] The stability of a composition can be determined by assessing its propensity to aggregate. Aggregation can be assessed by a number of non-limiting biochemical or biophysical techniques. For example, the aggregation of a composition can be evaluated using chromatography, for example, Size Exclusion Chromatography (SEC). SEC separates molecules based on size. A column is filled with semi-solid spheres of a polymeric gel that will admit ions and small molecules inside, but not large ones. When a protein composition is applied to the top of the column, the compactly folded proteins (i.e., non-aggregated proteins) distribute themselves through a larger volume of solvent than that available to the large protein aggregates. Consequently, the large aggregates move more rapidly through the column and, in this way, the mixture can be separated or fractionated into its components.Each fraction can be quantified separately (e.g., by light scattering) as it elutes from the gel. Thus, the % aggregation of a composition can be determined by comparing the concentration of a fraction with the total protein concentration applied to the gel. Stable compositions elute from the column essentially as a single fraction and show up essentially as a single peak in the elution profile or chromatogram. c) Binding affinity

[00182] The stability of a composition can be evaluated by determining its binding affinity to the target. A wide variety of methods for determining binding affinity are known in the art. An exemplary method for determining binding affinity employs surface plasmonic resonance. The resonance Petition 870260065699, dated 03 / 07 / 2026, page 93 / 532 86 / 250 surface plasmonics is an optical phenomenon that allows the analysis of biospecific interactions in real time by detecting changes in protein concentrations within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For more detailed descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[00183] In one aspect, the antigen-binding domain of the CAR comprises an amino acid sequence that is homologous to an antigen-binding domain amino acid sequence described herein, and the antigen-binding domain retains the desired functional properties of the anti-CD19 antibody fragments described herein. In a specific aspect, the CAR composition of the invention comprises an antibody fragment. In a further aspect, that antibody fragment comprises a scFv.

[00184] In several aspects, the antigen-binding domain of the CAR is constructed by modifying one or more amino acids within one or both of the variable regions (e.g., VH and / or VL), for example, within one or more CDR regions and / or within one or more framework regions. In one specific aspect, the CAR composition of the invention comprises an antibody fragment. In a further aspect, that antibody fragment comprises a scFv.

[00185] It will be understood by those skilled in the art that the antibody or antibody fragment of the invention may be further modified in such a way that it varies in amino acid sequence (e.g., from wild type), but not in the desired activity. For example, additional nucleotide substitutions may be made leading to amino acid substitutions at non-wild type amino acid residues. Petition 870260065699, dated 03 / 07 / 2026, p. 94 / 532 87 / 250 are essential to the protein. For example, a non-essential amino acid residue in a molecule can be replaced by another amino acid residue from the same side chain family. In another embodiment, an amino acid string can be replaced by a structurally similar string that differs in the order and / or composition of side chain family members; for example, a conservative substitution can be made, in which an amino acid residue is replaced by an amino acid residue with a similar side chain.

[00186] Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched beta side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[00187] Percentage identity, in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences that are identical. Two sequences are substantially identical if they both have a specified percentage of amino acid or nucleotide residues that are the same (e.g., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity along a specified region or, when unspecified, along the entire sequence), when compared and aligned for maximum match over a specified region. Petition 870260065699, dated 03 / 07 / 2026, p. 95 / 532 88 / 250 comparison window, or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, identity exists along a region with a length at least close to 50 nucleotides (or 10 amino acids) or, more preferably, along a region with a length of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids).

[00188] For sequence comparison, typically one sequence acts as a reference sequence to which sequences under test are compared. When using an algorithm for sequence comparison, the test and reference sequences are entered into a computer, subsequence coordinates are assigned, if necessary, and program sequence algorithm parameters are assigned. Standard program parameters may be used or alternative parameters may be assigned.The sequence comparison algorithm then calculates the percentage of sequence identity for the sequences under test with respect to the reference sequence, based on the program parameters. Sequence alignment methods for comparison are well known in the field. Optimal sequence alignment for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search using the similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology). Petition 870260065699, dated 03 / 07 / 2026, p. 96 / 532 89 / 250

[00189] Two examples of algorithms that are suitable for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. The software to perform the analyses with BLAST is publicly available through the National Center for Biotechnology Information.

[00190] The percentage of identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17, which was incorporated into the ALIGN program (version 2.0), using a PAM120 table of weighted values ​​for residues, a gap extension penalty of 12 and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453, which was incorporated into the GAP program of the GCG software package (available at www.gcg.com), using a Blossom 62 matrix or a PAM250 matrix and weighted gap values ​​of 16, 14, 12, 10, 8, 6 or 4 and weighted span values ​​of 1, 2, 3, 4, 5 or 6.

[00191] In one aspect, the present invention contemplates modifications of the initial amino acid sequence of the antibody or fragment (e.g., scFv) that generate functionally equivalent molecules. For example, the VH or VL of an antiCD19 binding domain, e.g., scFv, comprised in the CAR, can be modified to retain at least approximately 70%, 71%, 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the initial framework region of the VH or VL of the anti-CD19 binding domain, for example, scFv. The present invention Petition 870260065699, dated 03 / 07 / 2026, page 97 / 532 90 / 250 contemplates modifications to the entire CAR construct, for example, modifications to one or more amino acid sequences of the various domains of the constructed CAR in order to generate functionally equivalent molecules. The constructed CAR can be modified to retain at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the identity of the initially constructed CAR. Transmembrane domain

[00192] With respect to the transmembrane domain, in several embodiments, a CAR can be designed to comprise a transmembrane domain attached to the extracellular domain of the CAR. A transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids from the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids from the intracellular region). In one aspect, the transmembrane domain is the one that is associated with one of the other domains of the CAR used.In some cases, the transmembrane domain can be selected or modified by amino acid substitution to prevent the binding of such domains to the transmembrane domains of the same or different proteins on the membrane surface, for example, to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another CART on the cell surface. In a different aspect, the amino acid sequence of the transmembrane domain can be modified or... Petition 870260065699, dated 03 / 07 / 2026, page 98 / 532 91 / 250 replaced in order to minimize interactions with the native linking partner's linking domains present in the same CART.

[00193] The transmembrane domain can be derived from a natural or recombinant source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of, for example, the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[00194] In some cases, the transmembrane domain may be attached to the extracellular region of the CAR, for example, the antigen-binding domain of the CAR, via a link, for example, a link of a human protein. For example, in one embodiment, the link may be a human Ig (immunoglobulin) link, for example, an IgG4 link or a CD8a link. In one embodiment, the link or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:14. In one aspect, the transmembrane domain comprises (e.g., consists of) a transmembrane domain of SEQ ID NO:15.

[00195] In one aspect, the link or spacer comprises an IgG4 link. For example, in one embodiment, the link or spacer comprises an amino acid sequence link: ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM Petition 870260065699, dated 03 / 07 / 2026, page 99 / 532 92 / 250 TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO:45). In some embodiments, the articulation or spacer comprises an articulation encoded by a nucleotide sequence formed by GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGA GTTCTGGGGGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCA AGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTG GTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCC GGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTG ACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTG TAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCAT CAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACC CTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCT GACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGG AGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC CCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCG GCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTA GCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG AAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO:46).

[00196] In one aspect, the linkage or spacer comprises an IgD linkage. For example, in one embodiment, the linkage or spacer comprises an amino acid sequence linkage. RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTRGGGEEKKK EKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCF VVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLP RSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSD PPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPG Petition 870260065699, dated 03 / 07 / 2026, page 100 / 532 93 / 250 STTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYV TDH (SEQ ID NO:47). In some modalities,a joint or spacer comprises a joint encoded by a nucleotide sequence formed by AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACT GCACAGCCCCAGGCAGA AAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGGGAGACCAAG ACCCCTGAATGTCCATCCCCATACCCAGCCGCTGGGCGTCTATCTTC TTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACC TTTACATGTTTCGTCGTGGGCTCTGAAGGATGCCCATTTG ACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGA AGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACT CAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCT GTCACATGTACTCTAAATCATCCTAGCCTGCCCCGCCCT ATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAG CCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCT GGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGC TCATGTGGCTGGAGGACCAGCGGAGAGTGAACACAGCGGCTTC GCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATCTG GGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGC CAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCC TGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACTGACTGGAGGTTTCCTACGTGACTGACTGACTGGAGGAAGATAGCAGGACCCC TGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACTGACC (ID NO: 48).

[00197] In one aspect, the transmembrane domain may be recombinant, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In another aspect, a triplet of phenylalanine, tryptophan, and valine may be found at each end of a recombinant transmembrane domain. Petition 870260065699, dated 03 / 07 / 2026, p. 101 / 532 94 / 250

[00198] Optionally, a short linking oligo- or polypeptide, containing between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in one embodiment, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO:49). In some embodiments, the linker is encoded by a nucleotide sequence formed by GGTGGGCGAGGTCTTGGAGGTGGAGGTTCC (SEQ ID NO:50). Cytoplasmic domain

[00199] The cytoplasmic domain or region of the CAR includes an intracellular signaling domain. An intracellular signaling domain is generally responsible for activating at least one of the normal effector functions of the immune cell into which the CAR has been introduced. The term effector function refers to a specialized function of a cell. The effector function of a T cell, for example, may be cytolytic activity or helper activity, including cytokine secretion. Thus, the term intracellular signaling domain refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. Although normally the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire chain.To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain provided that it transduces the effector function signal. The term intracellular signaling domain, therefore, is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[00200] Examples of intracellular signaling domains for Petition 870260065699, dated 03 / 07 / 2026, page 102 / 532 95 / 250 uses in the CAR of the invention include cytoplasmic sequences of the T cell receptor (TCR) and coreceptors that act in synergy to initiate signal transduction after antigen-receptor interaction, as well as any derivative or variant of these sequences that have the same functional capacity.

[00201] It is known that signals generated through the TCR alone are insufficient for the full activation of T cells and that a secondary and / or co-stimulatory signal is also necessary. Thus, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act independently of the antigen to provide a secondary or co-stimulatory signal (secondary cytoplasmic domain, for example, a co-stimulatory domain).

[00202] A primary signaling domain regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as tyrosine-based immunoreceptor activating motifs or ITAMs.

[00203] Examples of ITAMs containing primary intracellular signaling domains that are of particular use in the invention include those of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, a CAR of the invention comprises an intracellular signaling domain, for example, a primary CD3-zeta signaling domain.

[00204] In one embodiment, a primary signaling domain comprises a modified ITAM domain, for example, a mutant ITAM domain with altered activity (e.g., increased or Petition 870260065699, dated 03 / 07 / 2026, page 103 / 532 96 / 250 diminished) when compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a primary intracellular signaling domain containing modified ITAM, for example, a primary intracellular signaling domain containing optimized and / or truncated ITAM. In one embodiment, a primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[00205] The intracellular signaling domain of the CAR may comprise the CD3-zeta signaling domain alone or may be combined with any desired intracellular signaling domain(s) useful in the context of a CAR of the invention. For example, the intracellular signaling domain of the CAR may comprise a portion of the CD3 zeta chain and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen or its ligands, that is required for an efficient lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7H3, and a ligand that specifically binds to CD83, and the like.For example, costimulation by CD27 has been shown to enhance the expansion, effector function, and survival of human CART cells in vitro and increases the persistence and antitumor activity in vivo of T cells (Song et al. Blood. 2012; 119(3):696-706).

[00206] The intracellular signaling sequences within the cytoplasmic portion of the CAR of the invention can be linked together randomly or in a specified order. Optionally, a short linking oligo- or polypeptide, for example, containing 2 and 10 Petition 870260065699, dated 03 / 07 / 2026, page 104 / 532 97 / 250 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length can form the link between the intracellular signaling sequence. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In another embodiment, a single amino acid, for example, an alanine or a glycine, can be used as a suitable linker.

[00207] In one aspect, the intracellular signaling domain is designed to comprise two or more, for example, 2, 3, 4, 5 or more co-stimulatory signaling domains. In one embodiment, the two or more, for example, 2, 3, 4, 5 or more co-stimulatory signaling domains, are separated by a linker molecule, for example, a linker molecule described herein. In one embodiment, the intracellular signaling domain comprises two co-stimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[00208] In one aspect, the intracellular signaling domain is created to encompass the CD3-zeta signaling domain and the CD28 signaling domain. In one aspect, the intracellular signaling domain is created to encompass the CD3-zeta signaling domain and the 4-1BB signaling domain. In one aspect, the 4-1BB signaling domain is a signaling domain of SEQ ID NO: 16. In one aspect, the CD3-zeta signaling domain is a signaling domain of SEQ ID NO: 17.

[00209] In one aspect, the intracellular signaling domain is designed to encompass the CD3-zeta signaling domain and the CD27 signaling domain. In one aspect, the CD27 signaling domain comprises an amino acid sequence formed by Petition 870260065699, dated 03 / 07 / 2026, page 105 / 532 98 / 250 QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACS P (SEQ ID NO:51). In one aspect, the CD27 signaling domain is encoded by a nucleic acid sequence formed by AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACAT GACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCT ATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO:52).

[00210] In one aspect, the cell expressing the CAR described herein may further comprise a second CAR, for example, a second CAR that includes a different antigen-binding domain, for example, against the same target (CD19) or a different target (for example, CD123). In another embodiment, when the cell expressing the CAR comprises two or more different CARs, the antigen-binding domains of the different CARs may be such that the antigen-binding domains do not interact with each other. For example, a cell expressing a first and a second CAR may have an antigen-binding domain of the first CAR, for example, in fragment form, for example, an scFv, which does not form an association with the antigen-binding domain of the second CAR, for example, the antigen-binding domain of the second CAR is a VHH.

[00211] In another aspect, the CAR-expressing cell described here may also express another agent, for example, an agent that enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, for example, PD1, may, in some embodiments, decrease the ability of a CAR-expressing cell to mount an effector immune response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, Petition 870260065699, dated 03 / 07 / 2026, page 106 / 532 99 / 250 for example, an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, for example, an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, for example, of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of these (for example, at least a portion of an extracellular domain of any of these), and a second polypeptide that is an intracellular signaling domain described herein (for example, comprising a co-stimulatory domain (for example, 41BB, CD27 or CD28, for example, as described herein) and / or a primary signaling domain (for example, a CD3 zeta signaling domain described herein)).In one embodiment, the agent comprises a first PD1 polypeptide, or a fragment thereof (e.g., at least a portion of an extracellular PD1 domain), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein). PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to negatively regulate T cell activation when bound to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:2817; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al.2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

[00212] In one modality, the agent understands the domain Petition 870260065699, dated 03 / 07 / 2026, page 107 / 532 100 / 250 extracellular (ECD) of an inhibitory molecule, for example, Programmed Cell Death protein 1 (PD1), which can be fused to a transmembrane domain and intracellular signaling domains such as 41BB and CD3 zeta (also referred to in this descriptive report as CAR PD1). In one embodiment, the CAR PD1, when used in combination with a CAR CD19 described herein, enhances T cell persistence. In one embodiment, the CAR is a CAR PD1 comprising the extracellular domain of PD1, indicated as underlined in SEQ ID NO: 121. In one embodiment, the CAR PD1 comprises the amino acid sequence of SEQ ID NO: 121.

[00213] Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsnt sesfvlnwyrmspsnqtdklaafpedrsqpgqdçrfrvtqlpngrdfhmsvvrarrndsgtylçga islapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpe acrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedg cscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkpr rknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO:121).

[00214] In one embodiment, CAR PD1 comprises the amino acid sequence provided below (SEQ ID NO:119).

[00215] pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqt dklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtYlcgaislapkaqjkeslraelrvt erraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfa cdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvk fsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkm aeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO:119).

[00216] In one embodiment, the agent comprises a nucleic acid sequence encoding the CAR PD1, for example, the CAR PD1 described herein. In one embodiment, the nucleic acid sequence for the CAR PD1 is shown below, with the ECD of PD1 underlined, in SEQ ID NO: 120 Petition 870260065699, dated 03 / 07 / 2026, p. 108 / 532 101 / 250

[00217] atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgcta qaccacccggatggtttctggactctccqgatcgcccgtggaatcccccaaccttctcaccggcac tcttqqttqtgactqagggcqataatgcgaccttcacgtgctcgttctccaacacctccqaatcattc qtqçtqaaçtqqtaççqçatqaqçç€qtçaaaç€aqaççqaçaιaqçtçqç€qçqtttç€qqaa qatcqgtcgcaaccgggacaggattgtcggttccgcgtgactcaactqccgaatgqcagagactt ccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctqtgcggagccatctcq ctqqcgcctaaggcccaaatcaaagagagcttgagggccgaactgaqagtgaccgagcgca qaqctgaggtgccaactgcacatccatccccatcgcctcggcctgcqqqgcagtttcagaccctq qtcacqaccactccggcgccgcgcccaccgactccggccccaactatcqcgagccagcccctq tcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggact tcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcat caccctgtactgcaagcggggtcggaaaaagcttctgtacattttcaagcagcccttcatgaggcc cgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggag gttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccaga accagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgt ataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggaga gcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaagga cacatacgatgccctgcacatgcaggcccttccccctcgc (SEQ ID NO: 120).

[00218] In another aspect, the present invention provides a population of cells expressing CARs, for example, CART cells. In some embodiments, the population of cells expressing CARs comprises a mixture of cells expressing different CARs. For example, in one embodiment, the CART cell population may include a first cell expressing a CAR containing an anti-CD19 binding domain described herein and a second cell expressing a CAR with a different anti-CD19 binding domain, for example, an anti-CD19 binding domain described herein that differs from the anti-CD19 binding domain in the CAR expressed by the first cell. As another example, the population of cells expressing CARs may include a Petition 870260065699, dated 03 / 07 / 2026, page 109 / 532 102 / 250 first cell expressing a CAR that includes an anti-CD19 binding domain, for example, as described herein, and a second cell expressing a CAR that includes an antigen-binding domain for a target other than CD19 (e.g., CD123). In one embodiment, the population of cells expressing CARs includes, for example, a first cell expressing a CAR that includes a primary intracellular signaling domain and a second cell expressing a CAR that includes a secondary signaling domain.

[00219] In another aspect, the present invention provides a population of cells, wherein at least one cell in the population expresses a CAR containing an anti-CD19 domain described herein and a second cell expressing another agent, for example, an agent that enhances the activity of a cell expressing a CAR. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, for example, may, in some embodiments, decrease the ability of a cell expressing a CAR to mount an effector immune response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, for example, an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, for example, an intracellular signaling domain described herein.In one embodiment, the agent comprises a first polypeptide, for example, of an inhibitory molecule such as PD1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta, or a fragment of any of these (for example, at least a portion of an extracellular domain of any of these), and a second polypeptide that is an intracellular signaling domain described herein (for example, comprising a co-stimulatory domain (e.g., 41BB, CD27). Petition 870260065699, dated 03 / 07 / 2026, page 110 / 532 103 / 250 or CD28, for example, as described herein) and / or a primary signaling domain (for example, a CD3 zeta signaling domain described herein)). In one embodiment, the agent comprises a first PD1 polypeptide, or a fragment thereof (for example, at least a portion of the extracellular PD1 domain), and a second polypeptide of an intracellular signaling domain described herein (for example, a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein). RNA transfection

[00220] The present invention describes methods for producing an RNA CAR transcribed in vitro. The present invention also includes an RNA construct encoding a CAR that can be transfected directly into a cell. One method for generating mRNA for use in transfection may involve in vitro transcription (IVT) of a template with specially designed primers, followed by the addition of poly A, to produce a construct containing untranslated sequences (UTRs) at 3' and 5', a 5' cap and / or an Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO:118). The RNA thus produced can be efficiently transfected into different cell types. In one aspect, the template includes sequences for the CAR.

[00221] In one aspect, the anti-CD19 CAR is encoded by messenger RNA (mRNA). In one aspect, the mRNA encoding the anti-CD19 CAR is introduced into a T cell for the production of a CART cell.

[00222] In one embodiment, the CAR of RNA transcribed in vitro can be introduced into a cell in the form of transient transfection. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). The DNA of interest, Petition 870260065699, dated 03 / 07 / 2026, p. 111 / 532 104 / 250 from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable DNA source. The desired template for in vitro transcription is a CAR of the present invention. For example, the template for the RNA CAR comprises an extracellular region containing a single-stranded variable domain of an anti-tumor antibody; a linking region, a transmembrane domain (e.g., a CD8a transmembrane domain); and a cytoplasmic region that includes an intracellular signaling domain, for example, comprising the CD3-zeta signaling domain and the 4-1BB signaling domain.

[00223] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA may be from a naturally occurring DNA sequence in an organism's genome. In one embodiment, the nucleic acid may include some or all of the 5' and / or 3' untranslated regions (UTRs). The nucleic acid may include exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence including 5' and 3' UTRs. The DNA may alternatively be an artificial DNA sequence that is not normally expressed in a natural organism. An exemplary artificial DNA sequence is one that contains gene fragments that are linked together to form an open reading frame encoding a fusion protein. The DNA fragments that are linked together may be from a single organism or from more than one organism.

[00224] PCR is used to generate a template for in vitro transcription of mRNA that is used for transfection. Methods Petition 870260065699, dated 03 / 07 / 2026, p. 112 / 532 105 / 250 primers for performing PCR are well known in the art. PCR primers are designed to contain regions that are substantially complementary to regions of the DNA to be used as a template for PCR. Substantially complementary, in this descriptive report, refers to nucleotide sequences in which most or all bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are capable of annealing or hybridizing with the intended target DNA under annealing conditions used for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, primers can be designed to amplify the part of a nucleic acid that is normally transcribed in cells (the open reading frame), including the 5' and 3' UTRs.Primers can also be designed to amplify a portion of a nucleic acid that encodes a specific domain of interest. In one embodiment, primers are designed to amplify the coding region of a human cDNA, including all or parts of the 5' and 3' UTRs. Useful primers for PCR can be generated by synthetic methods that are well known in the art. Forward primers are primers that contain a region of nucleotides that are substantially complementary to nucleotides in the upstream DNA template of the DNA sequence to be amplified. Upstream, in this descriptive report, refers to a location 5' for the DNA sequence to be amplified relative to the coding strand. Reverse primers are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that lies downstream of the DNA sequence to be amplified.Downstream, in this descriptive report, refers to a. Petition 870260065699, dated 03 / 07 / 2026, p. 113 / 532 106 / 250 location 3' for the DNA sequence to be amplified relative to the coding strand.

[00225] Any DNA polymerase useful for PCR can be used in the methods described herein. The reagents and polymerase are commercially available from a number of suppliers.

[00226] Chemical structures capable of promoting stability and / or translation efficiency can also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between one and 3000 nucleotides long. The length of the 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, the design of PCR primers that anneal to different regions of the UTRs. The use of this approach allows any person skilled in the art to modify the lengths of the 5' and 3' UTRs needed to achieve optimal translation efficiency after transfection of the transcribed RNA.

[00227] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other template modifications. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful to modify the stability and / or translation efficiency of RNA. For example, AU-rich elements in 3' UTR sequences are known to decrease mRNA stability. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs that are well known in the art.

[00228] In one embodiment, the 5' UTR may contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5' UTR that is not endogenous to the nucleic acid of interest is Petition 870260065699, dated 03 / 07 / 2026, page 114 / 532 107 / 250 added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the translation efficiency of some RNA transcripts, however, they do not appear to be necessary to enable the efficient translation of all RNAs. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5' UTR may be the 5' UTR of an RNA virus whose RNA-like genome is stable in cells. In other embodiments, various nucleotide analogs may be used in the 3' or 5' UTR to prevent degradation by exonucleases of the mRNA.

[00229] To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that acts as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter incorporates itself into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, among others, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3, and SP6 promoters are known in the art.

[00230] In a preferred embodiment, mRNA has a cap at the 5' end and a poly(A) tail at the 3' end that determine ribosome binding, translation initiation, and mRNA stability in the cell. In a circular DNA template, for example, plasmid DNA, RNA polymerase creates a long concatenated product that is not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end results in normal-sized mRNA that is not effective for transfection in eukaryotes even Petition 870260065699, dated 03 / 07 / 2026, page 115 / 532 108 / 250 if this is polyadenylated after transcription.

[00231] In a linear DNA template, the RNA polymerase of phage T7 is able to extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:622336 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[00232] The conventional method of integrating polyA / T segments into a DNA template is molecular cloning. However, the polyA / T sequence integrated into the plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time-consuming, but also frequently unreliable. This is why a method for constructing DNA templates with a 3' polyA / T segment without cloning is highly desirable.

[00233] The polyA / T segment of the DNA template for transcription can be produced during PCR using a reverse primer containing a polyT tail, such as a 100T tail (SEQ ID NO: 110) (the size can be 50-5000 T (SEQ ID NO: 111)), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also impart stability to RNAs and reduce their degradation. In general, the length of a poly(A) tail correlates positively with the stability of the transcribed RNA. In one embodiment, the poly(A) tail contains between 100 and 5000 adenosines (SEQ ID NO: 112).

[00234] Poly(A) RNA tails can be further extended after in vitro transcription using a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail by 100 nucleotides Petition 870260065699, dated 03 / 07 / 2026, p. 116 / 532 Increasing the number of nucleotides from 109 / 250 to between 300 and 400 (SEQ ID NO: 113) results in an almost twofold increase in RNA translation efficiency. Additionally, incorporating different chemical groups into the 3' end can increase mRNA stability. Such incorporation can involve modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase RNA stability.

[00235] 5' capping can also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods described here include a 5' cap. The 5' cap is introduced using techniques known in the subject and described (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[00236] The RNAs produced by the methods described herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal, or artificially designed sequence that initiates ribosome cap-independent binding to mRNA and facilitates translation initiation. Any solutes suitable for cell electroporation that may contain factors that facilitate permeability and cell viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants, may be included.

[00237] RNA can be introduced into target cells using any of a number of different methods, for example, commercially available methods which include, among others, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Petition 870260065699, dated 03 / 07 / 2026, p. 117 / 532 110 / 250 Germany), transfection mediated by cationic liposomes using lipofection, polymeric encapsulation, peptide-mediated transfection or biolytic systems for the release of particles such as gene guns (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[00238] Nucleic acid constructs encoding a CAR

[00239] The present invention also provides nucleic acid molecules encoding one or more CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

[00240] Thus, in one aspect, the invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD19 binding domain (e.g., a humanized anti-CD19 binding domain), a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, e.g., a co-stimulatory signaling domain and / or a primary signaling domain, e.g., a zeta chain. In one embodiment, the anti-CD19 binding domain is an anti-CD19 binding domain described herein, for example, an anti-CD19 binding domain comprising a sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, or a sequence with 95-99% identity to these.In one embodiment, the transmembrane domain is the transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment... Petition 870260065699, dated 03 / 07 / 2026, page 118 / 532 In one embodiment, the transmembrane domain comprises a sequence with SEQ ID NO: 15, or a sequence with 95-99% identity thereof. In one embodiment, the anti-CD19 binding domain is connected to the transmembrane domain by a splicing region, for example, a splicing region described herein. In one embodiment, the splicing region comprises SEQ ID NO:14, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:49, or a sequence with 95-99% identity thereof. In one embodiment, the isolated nucleic acid molecule further comprises a coding sequence for a costimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In one modality, the co-stimulatory domain comprises a sequence with SEQ ID NO:16, or a sequence with 95-99% identity to it.In one embodiment, the intracellular signaling domain comprises a 4-1BB functional signaling domain and a CD3 zeta functional signaling domain. In another embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence with 95-99% identity thereof, and the sequence SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence with 95-99% identity thereof, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

[00241] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence of SEQ ID NO: 13, an scFv domain with a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID Petition 870260065699, dated 03 / 07 / 2026, p. 119 / 532 112 / 250 NO:10, SEQ ID NO:11 and SEQ ID NO:12, (or a sequence with 95-99% identity to these), an articulation region of SEQ ID NO:14, SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:49 (or a sequence with 95-99% identity to this), a transmembrane domain with a sequence of SEQ ID NO:15 (or a sequence with 95-99% identity to this), a co-stimulatory domain of 4-1BB with a sequence of SEQ ID NO:16 or a co-stimulatory domain of CD27 with a sequence of SEQ ID NO:51 (or a sequence with 95-99% identity to these), and a stimulatory domain of CD3 with a sequence of SEQ ID NO:17 or SEQ ID NO:43 (or a sequence with 95-99% identity to these).

[00242] In another aspect, the invention relates to an isolated polypeptide molecule encoded by the nucleic acid molecule. In one embodiment, the isolated polypeptide molecule comprises a sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42 or a sequence with 95-99% identity thereof.

[00243] In another aspect, the invention relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule comprising an anti-CD19 binding domain, a transmembrane domain and an intracellular signaling domain containing a stimulatory domain, wherein said anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, or a sequence with 95-99% identity thereof.

[00244] In one embodiment, the CAR-encoded molecule Petition 870260065699, dated 03 / 07 / 2026, p. 120 / 532 113 / 250 comprises a coding sequence for a costimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). In one embodiment, the costimulatory domain comprises a sequence with SEQ ID NO:16. In one embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain comprises a sequence with SEQ ID NO:15. In one embodiment, the intracellular signaling domain comprises a 4-1BB functional signaling domain and a zeta functional signaling domain.In one embodiment, the intracellular signaling domain comprises the SEQ ID NO: 16 sequence and the SEQ ID NO: 17 sequence, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain. In one embodiment, the anti-CD19 binding domain is connected to the transmembrane domain by a linkage region. In one embodiment, the linkage region comprises SEQ ID NO: 14. In another embodiment, the linkage region comprises SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[00245] In another aspect, the invention relates to a CAR-encoded molecule comprising a leader sequence of SEQ ID NO: 13, an scFv domain with a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, or a Petition 870260065699, dated 03 / 07 / 2026, p. 121 / 532 114 / 250 sequence with 95-99% identity of these, an articulation region of SEQ ID NO:14, SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:49, a transmembrane domain with a sequence of SEQ ID NO:15, a 4-1BB co-stimulatory domain with a sequence of SEQ ID NO:16 or a CD27 co-stimulatory domain with a sequence of SEQ ID NO:51 and a CD3 zeta stimulatory domain with a sequence of SEQ ID NO:17 or SEQ ID NO:43. In one embodiment, the CAR-encoded molecule comprises a sequence selected from a group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, or a sequence with 95-99% identity to these.

[00246] The nucleic acid sequences encoding the desired molecules can be obtained using recombination methods known in the art, such as, for example, screening in libraries of cells expressing the gene, deriving the gene from a vector known to contain it, or direct isolation from cells and tissues containing it, using standard techniques. Alternatively, the gene of interest can be produced synthetically instead of being cloned.

[00247] The present invention also provides vectors into which DNA of the present invention is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for long-term gene transfer because they allow stable, durable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from oncoretroviruses, such as murine leukemia virus, in that they can achieve transduction in non-proliferating cells, such as hepatocytes. In addition, they have the added advantage of low immunogenicity. Petition 870260065699, dated 03 / 07 / 2026, page 122 / 532 115 / 250

[00248] In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5 / 35). In another embodiment, the expression of nucleic acids encoding CARs can be performed using transposons, such as the Sleeping Beauty system, Crisper, CAS9 and zinc finger nucleases. See below June et al. 2009 Nature Reviews Immunology 9.10: 704-716, incorporated herein by reference in this patent application.

[00249] In summary, the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operationally linking a nucleic acid encoding the CAR polypeptide or parts thereof to a promoter and incorporating the construct into an expression vector. Vectors may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[00250] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy, using a standard gene delivery protocol. The methods for gene delivery are known in the art. See, for example, U.S. Patents Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated in their entirety by reference in this patent application. In another embodiment, the invention provides a vector for gene therapy.

[00251] Nucleic acid can be cloned into a number of vector types. For example, nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of special interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Petition 870260065699, dated 03 / 07 / 2026, page 123 / 532 116 / 250

[00252] Furthermore, the expression vector can be delivered to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold (Spring Harbor Press, NY) and in other virology and molecular biology textbooks. Viruses that are useful as vectors include, among others, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, a suitable vector contains a functional origin of replication in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[00253] A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses offer a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the individual's cells either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Some adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[00254] Additional promoter elements, for example, enhancers, regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although some promoters have also been shown to contain functional elements located downstream of the start site. The spacing between promoter elements is often flexible, so the promoter's function is Petition 870260065699, dated 03 / 07 / 2026, page 124 / 532 117 / 250 preserved when elements are inverted or moved relative to each other. In the thymidine kinase (Tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements may apparently function cooperatively or independently to activate transcription.

[00255] An example of a promoter capable of expressing a CAR transgene in a mammalian T cell is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EF1a promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from cloned transgenes in a lentiviral vector. See, for example, Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). In one aspect, the EF1a promoter comprises the sequence provided as SEQ ID NO:100.

[00256] Another example of a promoter is the immediate and early promoter sequence of cytomegalovirus (CMV). This promoter sequence is a strong constitutive promoter sequence capable of directing high levels of expression of any polynucleotide sequence operationally linked to the promoter. However, other constitutive promoter sequences can also be used, including, among others, the early promoter of simian virus 40 (SV40), mouse mammary tumor virus (MMTV), the long terminal repeat (LTR) promoter of human immunodeficiency virus (HIV), the MoMuLV promoter, a promoter of avian leukemia virus, an immediate early promoter of Epstein-Barr virus, a promoter of Rous sarcoma virus, as well as promoters of human genes such as, among others, the actin promoter, the myosin promoter, the Petition 870260065699, dated 03 / 07 / 2026, page 125 / 532 118 / 250 elongation factor-α promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of initiating the expression of the polynucleotide sequence that is operationally switched on when such expression is desired, or stopping expression when expression is not desired. Examples of inducible promoters include, among others, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[00257] In order to evaluate the expression of a CAR polypeptide or parts thereof, the expression vector to be introduced into a cell may also contain a marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the cell population that is sought to be transfected or infected via viral vectors. In other respects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in host cells. Selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[00258] Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, for example, enzymatic activity. The expression of the reporter gene is evaluated at an appropriate time after Petition 870260065699, dated 03 / 07 / 2026, p. 126 / 532 119 / 250 that the DNA was introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., UiTei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be ligated to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

[00259] Methods for introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be rapidly introduced into a host cell, for example, a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the expression vector can be transferred to a host cell by physical, chemical, or biological means.

[00260] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for introducing a polynucleotide into a host cell is calcium phosphate-mediated transfection.

[00261] Biological methods for introducing a polynucleotide of interest into a host cell include the use of vectors. Petition 870260065699, dated 03 / 07 / 2026, page 127 / 532 120 / 250 DNA and RNA. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, for example, human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses and the like. See, for example, US Patents Nos. 5,350,674 and 5,585,362.

[00262] Chemical means of introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, spheres, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., a vesicle with an artificial membrane). Other cutting-edge techniques are available for nucleic acid delivery, such as targeted nanoparticle polynucleotide delivery or other submicrometer-sized delivery systems.

[00263] In the case of a non-viral delivery system being used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with a lipid can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linker molecule that is associated with the liposome and the oligonucleotide, enveloped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, or contained or complexed with a micelle or otherwise associated. Petition 870260065699, dated 03 / 07 / 2026, page 128 / 532 121 / 250 with a lipid. The compositions associated with lipids, lipid / DNA, or lipid / expression vector are not limited to any particular structure in solution. For example, the compositions may be present in a bilayer structure, such as micelles, or with a collapsed structure. Furthermore, they may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic lipids. For example, lipids include the fatty droplets that occur naturally in the cytoplasm, as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[00264] Lipids suitable for use can be obtained from commercial suppliers. For example, dimystil phosphatidylcholine (DMPC) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (DCP) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (Choi) can be obtained from Calbiochem-Behring; dimystil phosphatidylglycerol (DMPG) and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20 °C. Chloroform is used as the sole solvent because it evaporates more rapidly than methanol. Liposome is a generic term encompassing a variety of simple and multilayer lipid vehicles, formed by the generation of lipid bilayers or included aggregates. Liposomes can be characterized by their vesicular structures with a phospholipid bilayer membrane and an aqueous inner medium.Multilamellar liposomes have multiple lipid layers separated by an aqueous medium and form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components... Petition 870260065699, dated 03 / 07 / 2026, page 129 / 532 122 / 250 undergo autorearrangement before the formation of closed structures and trap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have solution structures different from the normal vesicular structure are also included. For example, lipids can assume a micellar structure or simply exist as non-uniform aggregates of lipid molecules. Additionally, lipofectamine-nucleic acid complexes are also considered.

[00265] Regardless of the method used to introduce exogenous nucleic acids into a host cell or to otherwise expose a cell to the inhibitor of the present invention, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell's DNA sequence. Such assays include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern Blotting, RT-PCR and PCR; biochemical assays, such as those that detect the presence or absence of a particular peptide, for example, by immunological means (ELISAs and Western blots) or by assays described herein to identify agents that fall within the scope of the invention.

[00266] The present invention further provides a vector comprising a nucleic acid molecule encoding a CAR. In one aspect, a CAR vector can be transduced directly into a cell, for example, a T cell. In one aspect, the vector is a cloning or expression vector, for example, a vector including, among others, one or more plasmids (for example, expression plasmids, cloning vectors, minicircles, tiny double chromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the constructed CAR in mammalian T cells. In one aspect, the mammalian T cell is a human T cell. Petition 870260065699, dated 03 / 07 / 2026, page 130 / 532 123 / 250 Sources of T cells

[00267] Prior to genetic expansion and modification, a source of T cells is obtained from an individual. The term individual is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of individuals include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, splenic tissue, and tumors. In certain aspects of the present invention, any number of T cell lines available in the art can be used. In certain aspects of the present invention, T cells can be obtained from a unit of blood collected from an individual using any number of techniques known to those skilled in the art, such as Ficoll™ separation.In a preferred embodiment, circulating blood cells from an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and placed in a buffer or appropriate medium for subsequent processing steps. In one aspect of the invention, the cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the washing solution does not contain calcium and may not contain magnesium or may not contain many, if not all, divalent cations. The initial activation steps in the absence of calcium may enhance activation. As those skilled in the art would readily recognize, a washing step can be performed by methods known to those skilled in the art, such as using a centrifuge. Petition 870260065699, dated 03 / 07 / 2026, page 131 / 532 124 / 250 semi-automatic direct flow cell processor (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other buffered or unbuffered saline solution. Alternatively, undesirable components of apheresis can be removed and the cells directly resuspended in the culture medium.

[00268] In one aspect, T cells are isolated from peripheral blood lymphocytes by lysis of red blood cells and depletion of monocytes, for example, by centrifugation through a PERCOLLT™ gradient or by countercurrent centrifugal elutriation. It is also possible to isolate a specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells, by positive or negative selection techniques. For example, in one aspect, T cells are isolated by incubation with anti-CD3 / anti-CD28 conjugated beads (e.g., 3x28), such as DYNABEADS® M-450 CD3 / CD28 T, for a period of time sufficient for positive selection of the desired T cells. In one aspect, the time period is approximately 30 minutes.In one additional aspect, the time period ranges from 30 minutes to 36 hours or longer, and includes all intermediate integer values. In another aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times can be used to isolate T cells in any situation where there are few T cells compared to other cell types, such as in the isolation of tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, the use of longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, Petition 870260065699, dated 03 / 07 / 2026, p. 132 / 532 By simply shortening or extending the time that T cells are allowed to bind to the CD3 / CD28 beads and / or increasing or decreasing the ratio of beads to T cells (as described in more detail below), subpopulations of T cells can be preferentially selected for or against early in the culture or at other times during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against early in the culture or at other desired times. Those skilled in the art would recognize that multiple rounds of selection can also be used in the context of this invention. In certain respects, it may be desirable to perform the selection procedure and use unselected cells in the activation and expansion process. Unselected cells can also be subjected to additional rounds of selection.

[00269] Enrichment of a T cell population by negative selection can be achieved with a combination of antibodies targeting surface markers unique to negatively selected cells. One method is cell separation and / or selection via negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies targeting cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLADR, and CD8. In certain respects, it may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain respects, regulatory T cells are depleted by spheres conjugated with anti-C25 or by another method. Petition 870260065699, dated 03 / 07 / 2026, page 133 / 532 126 / 250 similar selection.

[00270] In one embodiment, a population of T cells expressing one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B and perforin or other appropriate molecules, for example, other cytokines, can be selected. Methods for screening cell expression can be determined, for example, by the methods described in PCT Publication No. WO 2013 / 126712.

[00271] For the isolation of a desired population of cells by positive or negative selection, the concentration of cells and the surface area (e.g., particles such as spheres) can be varied. In certain aspects, it may be desirable to significantly decrease the volume in which the spheres and cells are mixed together (e.g., increase the cell concentration) to ensure maximum contact between cells and spheres. For example, in one aspect, a concentration of 2 billion cells / mL is used. In another aspect, a concentration of 1 billion cells / mL is used. In a further aspect, concentrations above 100 million cells / mL are used. In yet another aspect, a cell concentration equal to 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL is used. In one further aspect, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells / mL is used.In additional aspects, concentrations of 125 or 150 million cells / mL can be used. The use of high concentrations can result in higher cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative cells, or samples in which there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and... Petition 870260065699, dated 03 / 07 / 2026, page 134 / 532 127 / 250 would be desirable. For example, using a high cell concentration allows for more efficient selection of CD8+ T cells, which typically exhibit weaker CD28 expression.

[00272] In a related aspect, it may be desirable to use lower cell concentrations. Significantly diluting the T cell and surface mixture (e.g., particles such as spheres) allows minimizing interactions between the particles and the cells. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are captured more efficiently than CD8+ T cells at diluted concentrations. In one aspect, a used cell concentration is 5 x 10⁶ / mL. In other aspects, the used concentration may be approximately 1 x 10⁵ / mL to 1 x 10⁶ / mL, and any intermediate integer value.

[00273] In other respects, cells can be incubated in a centrifuge for varying periods of time at varying speeds, either at 2-10°C or at room temperature.

[00274] T cells for stimulation can also be frozen after a washing step. Without intending to be bound by theory, the subsequent freeze-thaw step provides a more uniform product by removing granulocytes and, to some extent, monocytes from the cell population. After the washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% Dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or Plasmalyte-A 31.25%, 5% dextrose, 0.45% NaCl, Dextran 40 Petition 870260065699, dated 03 / 07 / 2026, page 135 / 532 128 / 250 10% and 5% dextrose. Dextrose, 20% human serum albumin and 7.5% DMSO or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a storage tank containing liquid nitrogen. Other controlled freezing methods may be used, as well as uncontrolled thawing immediately at -20°C or in liquid nitrogen.

[00275] In certain aspects, the cryopreserved cells are thawed and washed as described herein and left to stand for one hour at room temperature before activation using the methods of the present invention.

[00276] Additionally, the invention also contemplates the collection of blood cells or the product of apheresis from an individual at a time prior to when the expanded cells as described herein might be needed. Thus, the collection of cells to be expanded at the source can be carried out at any time point necessary, and the desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that have benefited from T cell therapy, such as those described herein. In one aspect, the blood sample collected or obtained by apheresis comes from a generally healthy individual who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for future use. In certain aspects, the T cells can be expanded, frozen, and used at a later time.In certain aspects, samples are collected from a patient immediately after a specific illness, as described here, but before any treatments. In an additional aspect, the cells are... Petition 870260065699, dated 03 / 07 / 2026, page 136 / 532 129 / 250 isolated from a blood sample collected or obtained by apheresis from an individual prior to any number of relevant therapeutic modalities, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506 antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, Cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and irradiation.

[00277] In a further aspect of the present invention, T cells are obtained from a patient directly after treatment that leaves the individual with functional T cells. In this regard, it has been observed that, after certain cancer treatments, especially treatments with drugs that cause damage to the immune system, immediately after treatment, during the period when patients would normally be recovering from treatment, the quality of T cells obtained can be optimal or improved in relation to their ability to expand ex vivo. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced grafting and in vivo expansion. Thus, in the context of the present invention, it is contemplated to collect blood cells, including T cells, dendritic cells or other cells of the hematopoietic lineage, during this recovery phase.Furthermore, in certain aspects, mobilization schemes (e.g., mobilization with GM-CSF) and conditioning can be employed to create a condition in an individual where the repopulation, regeneration, and / or expansion of specific cell types are favored, particularly during a defined time window after therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other immune system cells. Petition 870260065699, dated 03 / 07 / 2026, page 137 / 532 130 / 250 T cell activation and expansion

[00278] T cells can be activated and expanded generally using methods as described, for example, in US 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 US Patent Application Publication No. 20060121005.

[00279] In general, the T cells of the invention can be expanded by contact with a surface to which an agent that stimulates a signal associated with the CD3 / TCR complex and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells has been attached. Specifically, populations of T cells can be stimulated as described herein, such as by contact with an anti-CD3 antibody, or its antigen-binding fragment, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under appropriate conditions to stimulate T cell proliferation.To stimulate the proliferation of CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody are used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France), which can be used as can other methods commonly known in the technique (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999; Garland et al., J. Immunol Meth.

[00280] In certain aspects, the primary stimulatory signal and the co-stimulatory signal for the T cell can be provided by protocols Petition 870260065699, dated 03 / 07 / 2026, p. 138 / 532 131 / 250 different. For example, the agents delivering each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in cis formation) or to separate surfaces (i.e., in trans formation). Alternatively, one agent may be coupled to a surface and the other may be in solution. In one aspect, the co-stimulatory signal-delivering agent is bound to a cell surface and the primary activation signal-delivering agent is in solution or coupled to a surface. In certain aspects, both agents may be in solution. In one aspect, the agents may be in soluble form and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent that will bind to the agents. In this respect, see, for example, the US Patent Application Publications.Nos. 20040101519 and 20060034810 for artificial antigen-presenting cells (aAPCs), whose use is contemplated in the present invention to activate and expand T cells.

[00281] In one aspect, the two agents are immobilized on spheres, either on the same sphere, i.e., cis, or on separate spheres, i.e., trans. For example, the primary activation signal-providing agent is an anti-CD3 antibody, or its antigen-binding fragment, and the co-stimulatory signal-providing agent is an anti-CD28 antibody or its antigen-binding fragment; and the two agents are immobilized together on the same sphere in equimolar amounts. In one aspect, a 1:1 ratio is used for each antibody bound to the spheres for CD4+ T cell expansion and T cell growth. In certain aspects of the present invention, the ratio of antibodies against CD3:CD28 bound to the spheres is such that an increase in T cell expansion is observed when compared to the expansion observed using a 1:1 ratio. In one aspect Petition 870260065699, dated 03 / 07 / 2026, page 139 / 532 Specifically, an increase of approximately 1 to approximately 3 times is observed compared to the expansion observed using a 1:1 ratio. In one aspect, the ratio of anti-CD3:CD28 antibody bound to the beads varies from 100:1 to 1:100 and all intermediate integers in the range. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the CD3:CD28 ratio is less than one. In certain aspects of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In a particular aspect, a 1:100 ratio of anti-CD3:CD28 antibodies bound to beads is used. In one aspect, a 1:75 ratio of anti-CD3:CD28 antibodies bound to beads is used. In a further aspect, a 1:50 ratio of anti-CD3:CD28 antibodies bound to beads is used. In one aspect, a 1:30 ratio of antibodies against CD3:CD28 bound to beads is used.In a preferred aspect, a 1:10 ratio of antibodies against CD3:CD28 bound to beads is used. In another aspect, a 1:3 ratio of antibodies against CD3:CD28 bound to beads is used. In yet another aspect, a 3:1 ratio of antibodies against CD3:CD28 bound to beads is used.

[00282] Particle-to-cell ratios of 1:500 to 500:1 and any intermediate integer values ​​may be used to stimulate T cells or other target cells. As those skilled in the art can readily recognize, the particle-to-cell ratio may depend on the size of the particles relative to the target cell. For example, small spheres might bind to only a few cells, while larger spheres would bind to many. In certain respects, the cell-to-particle ratio ranges from 1:100 to 100:1 and any intermediate values, and in further respects, to stimulate T cells, a ratio of 1:9 to 9:1, as well as any intermediate values, may be used. The ratio Petition 870260065699, dated 03 / 07 / 2026, page 140 / 532 The ratio of anti-CD3 to anti-CD28 particles coupled to T cells that results in T cell stimulation can vary as noted above; however, certain preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with a preferred ratio of at least 1:1 particles per T cell. In one aspect, a particle-to-T cell ratio of 1:1 or less is used. In a special aspect, a preferred particle-to-cell ratio is 1:5. In additional aspects, the particle-to-cell ratio can be varied depending on the day of stimulation. For example, in one aspect, the particle-to-cell ratio is 1:1 to 10:1 on the first day, and additional particles are added to the cells every day or on alternate days thereafter for up to 10 days, at final ratios of 1:1 to 1:10 (based on cell counts on the day of addition).In one particular aspect, the particle-to-cell ratio is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In another aspect, particles are added daily or on alternate days to a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. In another aspect, the particle-to-cell ratio is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In another aspect, particles are added daily or on alternate days to a final ratio of 1:1 on the first day and 1:10 on the third and fifth days of stimulation. Those skilled in the art will recognize that a variety of other ratios may be suitable for use in the present invention. Specifically, the ratios will vary depending on the particle size and the cell size and type. In another aspect, the most typical ratios for use are in the vicinity of 1:1, 2:1, and 3:1 on the first day.

[00283] In further aspects of the present invention, the cells, Petition 870260065699, dated 03 / 07 / 2026, page 141 / 532 134 / 250 such as T cells, are combined with agent-coated beads, the beads and cells are subsequently separated and then cultured. In an alternative aspect, prior to culture, the agent-coated beads and cells are not separated but cultured together. In a further aspect, the beads and cells are first concentrated by applying a force, such as magnetic force, resulting in increased binding of markers on the cell surface, thus inducing cell stimulation.

[00284] By way of example, cell surface proteins can be linked, allowing the paramagnetic beads to which anti-CD3 and anti-CD28 antibodies are attached (3x28 beads) to contact T cells. In one aspect, the cells (e.g., 10⁴ to 10⁹ T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads at a 1:1 ratio) are combined in a buffer, e.g., PBS (without divalent cations such as calcium and magnesium). Again, those skilled in the art can readily recognize that any concentration of cells can be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample, or the entire sample (i.e., 100%) may comprise the target cell of interest. Thus, any number of cells is covered by the context of the present invention.In certain aspects, it may be desirable to significantly decrease the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum cell-particle contact. For example, in one aspect, a concentration close to 2 billion cells / mL is used. In another aspect, more than 100 million cells / mL is used. In a further aspect, a cell concentration equal to 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells / mL is used. In yet another aspect, a cell concentration starting from 75, 80, 85, 90, 95 or 100 million cells / mL is used. Petition 870260065699, dated 03 / 07 / 2026, p. 142 / 532 A concentration of 135 / 250 million cells / mL is used. In addition, concentrations of 125 or 150 million cells / mL can be used. The use of higher concentrations can result in greater cell yield, activation, and expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigens of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and, in certain aspects, it would be desirable to obtain them. For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[00285] In one aspect of the present invention, the mixture can be cultured for several hours (approximately 3 hours) up to approximately 14 days or any intermediate whole hour value. In one aspect, the mixture can be cultured for 21 days. In one aspect of the invention, the spheres and T cells are cultured together for approximately eight days. In one aspect, the spheres and T cells are cultured together for 2-3 days. Several stimulation cycles may also be desired, such that the T cell culture time can be equal to or greater than 60 days.Appropriate conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Media 1640 or X-vivo 15 (Lonza)) that may contain the factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFe and TNF-α or any other cell growth enhancers known to those skilled in the art. Other cell growth enhancers include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The means may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, XPetição 870260065699, dated 03 / 07 / 2026, page 143 / 532. 136 / 250 Vivo 15 and X-Vivo 20, Optimizer, added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or a sufficient amount of cytokine(s) for T cell growth and expansion. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cell cultures intended for infusion into an individual. Target cells are maintained under conditions necessary to support growth, e.g., appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[00286] T cells that have been exposed to stimulation for varying lengths of time may exhibit different characteristics. For example, typical blood derivatives or peripheral blood mononuclear cells obtained by apheresis have a population of helper T cells (TH, CD4+) that is higher than the population of cytotoxic or suppressor T cells (TC, CD8+). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors produces a T cell population, before approximately days 8-9, consisting predominantly of TH cells, while after approximately 8-9 days, the T cell population comprises an increasingly larger population of TC cells. Thus, depending on the therapeutic objective, infusing a T cell population comprising predominantly TH cells into an individual may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated, it may be beneficial to expand that subset to a greater degree.

[00287] Additionally, besides markers for CD4 and CD8, other phenotype markers vary, but largely in terms of reproducibility during the expansion process cycle. Petition 870260065699, dated 03 / 07 / 2026, page 144 / 532 137 / 250 cellular. Thus, such reproducibility makes it possible to adapt an activated T-cell product for specific purposes.

[00288] Once a CD19 CAR is constructed, several assays can be used to evaluate the molecule's activity, such as, among others, the ability to expand T cells after antigen stimulation, to sustain cell expansion in the absence of further stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of a CD19 CAR are described in more detail below.

[00289] Western blotting analysis of CAR expression in primary T cells can be employed to detect the presence of monomers and dimers. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1:1 mixture of CD4+ and CD8+ T cells) expressing CARs are expanded in vitro for more than 10 days, followed by lysis and SDS-PAGE under reducing conditions. CARs containing the complete cytoplasmic TCR-ζ domain and the endogenous TCR-ζ chain are detected by Western blotting using an antibody against the TCR-ζ chain. The same T cell sets are used for SDS-PAGE analysis under non-reducing conditions to allow assessment of covalent dimer formation.

[00290] The in vitro expansion of CAR+ T cells after antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4+ and CD8+ T cells is stimulated with aCD3 / aCD28 aAPCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include the CMV IE gene, the EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescence is evaluated on Day 6 of culture in CD4+ and / or CD8+ T cell subsets by flow cytometry. See, for example, Milone et al., Petition 870260065699, dated 03 / 07 / 2026, page 145 / 532 138 / 250 Molecular Therapy 17(8): 1453-1464 (2009). Alternatively, a mixture of CD4+ and CD8+ T cells is stimulated with aCD3 / aCD28-coated magnetic beads on Day 0 and transduced with CAR on Day 1 via a bicistronic lentiviral vector expressing CAR along with eGFP using a ribosomal skipping sequence 2A. Cultures are restimulated with K562 CD19+ cells (K562-CD19), wild-type K562 cells (K562 wild-type), or K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and anti-CD28 antibody (K562-BBL-3 / 28) after washing. Exogenous IL-2 is added to the cultures on alternate days at 100 IU / mL. GFP+ T cells are enumerated by flow cytometry using bead-based counting. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).

[00291] The sustained expansion of CAR+ T cells in the absence of repeated stimulation can also be measured. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). In summary, the average T cell volume (fl) is measured on Day 8 of culture using a Coulter Multisizer III particle counter, followed by stimulation with aCD3 / aCD28-coated magnetic particles on Day 0 and transduction with the indicated CAR on Day 1.

[00292] Animal models can also be used to assess CART activity. For example, the xenograft model using human CD19-specific CAR+ T cells can be used to treat primary human B-cell ALL in immunodeficient mice. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, after the establishment of ALL, mice are randomized to treatment groups. Different numbers of T cells constructed with αCD19-ζ and αCD19-BB-ζ are injected together, in a 1:1 ratio, into NOD-SCID-y- / -B-ALL mice. The number of Petition 870260065699, dated 03 / 07 / 2026, p. 146 / 532 139 / 250 copies of the aCD19-Z and aCD19-BB-Z vector in the spleen DNA of mice are evaluated at various time points after T cell injection. Animals are assessed for the presence of leukemia at weekly intervals. CD19+ blast cell counts of B-cell ALL are measured in the peripheral blood of mice injected with aCD19-Z CAR+ T cells or sham-transduced T cells. Survival curves for the groups are compared using the Log-Rank test. In addition, absolute CD4+ and CD8+ T cell counts in peripheral blood after 4 weeks of T cell injection in NOD-SCID-y- / - mice can also be analyzed. Mice are injected with leukemic cells and, 3 weeks later, injected with T cells constructed to express CAR by a bicistronic lentiviral vector encoding eGFP-bound CAR.T cells are normalized to 45-50% GFP+ T cells by mixing with sham-transduced cells before injection, and confirmed by flow cytometry. Animals are assessed for leukemia at 1-week intervals. Survival curves for the CAR+ T cell groups are compared using the Log-Rank test.

[00293] Dose-dependent response to CAR treatment can be assessed. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For example, peripheral blood is obtained 35-70 days after the establishment of leukemia in mice injected, on Day 21, with CAR T cells, an equivalent number of sham-transduced T cells, or no T cells. Mice from each group are randomly bled to determine peripheral blood CD19+ ALL blast counts and then sacrificed on Days 35 and 49. The remaining animals are evaluated on Days 57 and 70.

[00294] Assessment of cell proliferation and cytokine production Petition 870260065699, dated 03 / 07 / 2026, page 147 / 532 140 / 250 was previously described, for example, in Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, the assessment of CAR-mediated proliferation is performed in microtiter plates, mixing washed T cells with K562 cells expressing CD19 (K19) or CD32 and CD137 (KT32-BBL) to a final T cell:K562 ratio of 2:1. K562 cells are subjected to gamma radiation before use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to KT32-BBL cell cultures to serve as a positive control for T cell proliferation stimulation, as these signals support prolonged ex vivo expansion of CD8+ T cells. T cells are enumerated in cultures using CountBright™ fluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry as described by the manufacturer. CAR+ T cells are identified by GFP expression using T cells constructed with lentiviral vectors expressing CAR bound to eGFP-2A.For CAR+ T cells that do not express GFP, CAR+ T cells are detected with recombinant biotinylated C19 protein and an avidin-PE secondary conjugate. CD4+ and CD8+ expression on T cells is also detected simultaneously with specific monoclonal antibodies (BD Biosciences). Cytokines are measured in supernatants collected 24 hours after restimulation using the Human TH1 / TH2 Cytokine Detection Bead Array Kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. Fluorescence is assessed with a FACScalibur flow cytometer, and data are analyzed according to the manufacturer's instructions.

[00295] Cytotoxicity can be assessed by a standard 51Cr release assay. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, target cells (K562 cell lines and primary pro-B-ALL cells) are loaded with 51Cr (in the form of NaCrO4, New England Nuclear, Boston, MA) at 37°C for 2 hours with Petition 870260065699, dated 03 / 07 / 2026, page 148 / 532 141 / 250 cells are frequently agitated, washed twice in complete RPMI medium, and seeded onto microtiter plates. Effector T cells are mixed with target cells in wells in complete RPMI medium, at varying effector / target cell ratios (E:T). Additional cells containing medium only (spontaneous release, SR) or in a 1% solution of Triton-X 100 detergent (total release, TR) are also prepared. After 4 hours of incubation at 37°C, the supernatant from each well is collected. The amount of 51Cr released is then measured with a gamma particle counter (Packard Instrument Co., Waltham, MA). Each condition is reproduced and tested at least three times, and the percentage of lysis is calculated using the formula: % lysis = (ER - SR) / (TR - SR), where ER represents the average amount of 51Cr released for each experimental condition.

[00296] Imaging technologies can be employed to evaluate the specific transit and proliferation of CARs in tumor-bearing animal models. Such assays have been described, for example, in Barrett et al., Human Gene Therapy 22:1575-1586 (2011). Briefly, NOD / SCID / yc- / -(NSG) mice are injected intravenously with Nalm-6 cells, followed, 7 days later, by the injection of T cells 4 hours after electroporation with CAR constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and images of the mice are obtained for bioluminescence detection.Alternatively, the therapeutic efficacy and specificity of a single injection of CAR+ T cells in the Nalm-6 xenograft model can be assessed as follows: NSG mice are injected with Nalm-6 transduced to stably express mouse luciferase, followed, seven days later, by a single injection into the tail vein of electroporated CAR CD19 T cells. Images of the animals are obtained at various time points after the injection. Petition 870260065699, dated 03 / 07 / 2026, page 149 / 532 142 / 250 injection. For example, heat photon density maps of firefly luciferase-positive leukemia in representative mice can be generated on Day 5 (2 days before treatment) and Day 8 (24 hours after CAR+ PBLs).

[00297] Other tests, including those in the Examples section below, as well as those known in the art, may also be used to evaluate the CAR CD19 conductivity of the invention. Therapeutic application Diseases and / or disorders associated with CD19

[00298] In one aspect, the invention provides methods for treating a disease associated with CD19 expression. In one aspect, the invention provides methods for treating a disease in which part of the tumor is CD19 negative and part of the tumor is CD19 positive. For example, the CAR of the invention is useful for treating individuals who have undergone treatment for a disease associated with elevated CD19 expression, where the individual undergoing treatment for elevated CD19 levels exhibits a disease associated with elevated CD19 levels.

[00299] In one aspect, the invention relates to a vector comprising a CD19 CAR operatively linked to a promoter for expression in mammalian T cells. In one aspect, the invention provides a recombinant T cell expressing the CD19 CAR for use in the treatment of CD19-expressing tumors, wherein the recombinant T cell expressing the CD19 CAR is referred to as CD19 CART. In one aspect, the CD19 CART of the invention is capable of contacting a tumor cell with at least one CD19 CAR of the invention expressed on its surface, such that the CART is targeted to the tumor cell and tumor growth is inhibited.

[00300] In one aspect, the invention relates to a method for inhibiting the growth of a tumor cell expressing CD19, Petition 870260065699, dated 03 / 07 / 2026, page 150 / 532 143 / 250 comprising contacting the tumor cell with a CD19 CART cell of the present invention such that the CART is activated in response to the antigen and directed to the cancer cell, wherein tumor growth is inhibited.

[00301] In one aspect, the invention relates to a method for treating cancer in an individual. The method comprises administering to the individual a CD19 CART cell of the present invention, such that the cancer is treated in the individual. An example of cancer that is treatable by the CD19 CART cell of the invention is a cancer associated with CD19 expression. In one aspect, the cancer associated with CD19 expression is a hematological cancer. In one aspect, the hematological cancer is a leukemia or lymphoma. In one aspect, a cancer associated with CD19 expression includes cancers and malignancies including, among others, for example, one or more acute leukemias including, among others, for example, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including, but not limited to, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL).Additional cancers or hematological conditions associated with CD19 expression include, among others, for example, prolymphocytic B-cell leukemia, plasmacytoid dendritic cell blast neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and preleukemia, which represents a diverse collection of hematological conditions. Petition 870260065699, dated 03 / 07 / 2026, page 151 / 532 144 / 250 united by ineffective production (or dysplasia) of myeloid and similar blood cells. Furthermore, a disease associated with CD19 expression includes, among others, for example, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with CD19 expression.

[00302] In some embodiments, a cancer that can be treated with a CD19 CAR, for example, as described herein, is multiple myeloma. Multiple myeloma is a blood cancer characterized by the accumulation of a plasma cell clone in the bone marrow. Current therapies for multiple myeloma include, among others, treatment with lenalidomide, which is an analogue of thalidomide. Lenalidomide has activities that include antitumor activity, angiogenesis inhibition, and immunomodulation. In general, myeloma cells are believed to be negative for CD19 expression by flow cytometry. The present invention encompasses the recognition that a small percentage of myeloma tumor cells express CD19, as demonstrated in Example 6. Thus, in some embodiments, a CD19 CAR, for example, as described herein, can be used to target myeloma cells.In some modalities, CAR CD19 therapy can be used in combination with one or more additional therapies, for example, treatment with lenalidomide.

[00303] The invention includes a type of cell therapy in which T cells are genetically modified to express a chimeric antigen receptor (CAR) and the CAR T cell is infused into a recipient in need of this treatment. The infused cell is able to eliminate tumor cells in the recipient. Unlike antibody therapies, CAR-modified T cells are able to replicate in vivo resulting in prolonged persistence that can lead to sustained tumor control. In several respects, the T cells administered to the patient, or the progeny of these cells, persist in Petition 870260065699, dated 03 / 07 / 2026, page 152 / 532 145 / 250 patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years or five years after T cell administration to the patient.

[00304] The invention also includes a type of cell therapy in which T cells are modified, for example, by RNA transcribed in vitro, to transiently express a chimeric antigen receptor (CAR), and the CAR T cell is infused into the recipient in need of this treatment. The infused cell is able to eliminate tumor cells in the recipient. Thus, in several respects, the T cells administered to the patient are present for less than a month, for example, three weeks, two weeks, one week, after the administration of the T cell to the patient.

[00305] Without intending to adhere to any particular theory, the antitumor immune response elicited by CAR-modified T cells may be an active or passive immune response, or alternatively, it may be due to a direct versus indirect immune response. In one aspect, CAR-transduced T cells exhibit secretion of specific pro-inflammatory cytokines and potent cytolytic activity in response to human cancer cells expressing CD19, resist inhibition by soluble CD19, act as mediators in cell death by the bystander effect, and in the regression of an established human tumor. For example, tumor cells with less antigen within a heterogeneous tumor field expressing CD19 may be susceptible to indirect destruction by CD19-redirected T cells that had previously reacted against adjacent antigen-positive cancer cells. Petition 870260065699, dated 03 / 07 / 2026, page 153 / 532 146 / 250

[00306] In one aspect, the CAR-modified wholly human T cells of the invention can be a type of vaccine for ex vivo immunization and / or in vivo therapy in a mammal. In one aspect, the mammal is a human being.

[00307] With respect to ex vivo immunization, at least one of the following occurs in vitro before administration of the cell to a mammal: i) cell expansion, ii) introduction of a nucleic acid encoding a CAR into the cells, or iii) cryopreservation of the cells.

[00308] Ex vivo procedures are well known in the art and discussed in more detail below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR described herein. The CAR-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the recipient.

[00309] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Patent No. 5,199,942, incorporated herein by reference in this patent application, and may be applied to the cells of the present invention. Other suitable methods are known in the art, therefore the present invention is not limited to any particular method of ex vivo cell expansion. Briefly, the ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells, belonging to a mammal, from peripheral blood or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cell growth factors described in U.S. Patent No. 5,199,942, other factors, such as flt3-L, IL-1, IL-3 and the c-kit ligand, may be Petition 870260065699, dated 03 / 07 / 2026, page 154 / 532 147 / 250 used for cell cultivation and expansion.

[00310] In addition to the use of a cellular vaccine in terms of ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization in order to elicit a targeted immune response against an antigen in a patient.

[00311] Generally, activated and expanded cells, as described herein, can be used in the treatment and prevention of diseases arising in immunocompromised individuals. Specifically, the CAR-modified cells of the invention are used in the treatment of diseases, disorders, and conditions associated with CD19 expression. In certain respects, the cells of the invention are used in the treatment of patients at risk of developing diseases, disorders, and conditions associated with CD19 expression. Thus, the present invention provides methods for the treatment or prevention of diseases, disorders, and conditions associated with CD19 expression, comprising administering to an individual in need of such treatment a therapeutically effective quantity of the CAR-modified cells of the invention.

[00312] In one aspect, the CART cells of the invention can be used to treat a proliferative disease such as cancer or malignancy or a precancerous condition such as myelodysplasia, myelodysplastic syndrome or pre-leukemia. In one aspect, the cancer is a hematological cancer. In one aspect, the hematological cancer is leukemia or lymphoma. In one aspect, the CART cells of the invention can be used to treat cancers and malignancies such as, among others, for example, acute leukemias including, among others, for example, acute lymphoblastic leukemia of B cells (B-ALL), acute lymphoblastic leukemia of T cells (T-ALL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including, among others, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL);. Petition 870260065699, dated 03 / 07 / 2026, page 155 / 532 148 / 250 additional hematological cancers or conditions including, but not limited to, for example, prolymphocytic B-cell leukemia, plasmacytoid dendritic cell blast neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia and preleukemia, which represents a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid or similar blood cells.Furthermore, diseases associated with CD19 expression include, among others, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases that express CD19. Non-cancer-related indications associated with CD19 expression include, among others, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplants.

[00313] The CAR-modified T cells of the present invention can be administered alone or in the form of a pharmaceutical composition in combination with diluents and / or other components such as IL-2 or other cytokines or cell populations. Hematologic cancer

[00314] Cancerous hematological conditions are types of cancer, such as leukemia, and malignant lymphoproliferative conditions that affect the blood, bone marrow, and lymphatic system.

[00315] Leukemia can be classified as acute leukemia and chronic leukemia. Acute leukemia can be further classified as acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL). A Petition 870260065699, dated 03 / 07 / 2026, page 156 / 532 149 / 250 Chronic leukemia includes chronic myelogenous leukemia (CML) and chronic lymphocytic leukemia (CLL). Other related conditions include myelodysplastic syndromes (MDS, formerly known as pre-leukemia) which represent a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells and risk of transformation to AML.

[00316] The present invention provides compositions and methods for treating cancer. In one aspect, cancer is a hematological cancer including, among others, leukemia or lymphoma. In one aspect, the CART cells of the invention can be used to treat cancers and malignancies such as, among others, for example, acute leukemias including, among others, for example, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including, among others, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL);Hematologic cancers or additional hematologic conditions including, but not limited to, for example, prolymphocytic B-cell leukemia, plasmacytoid dendritic cell blast neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia and preleukemia, which represents a diverse collection of hematologic conditions united by ineffective production (or dysplasia) of myeloid and similar blood cells. Furthermore, a disease associated with CD19 expression includes, among others, for example, atypical and / or non-classical cancers, malignancies; Petition 870260065699, dated 03 / 07 / 2026, p. 157 / 532 150 / 250 precancerous conditions or proliferative diseases that express CD19.

[00317] The present invention also provides methods for inhibiting the proliferation or reducing a population of cells expressing CD19, wherein the methods comprise contacting a population of cells, comprising a cell expressing CD19, with an anti-CD19 CART cell of the invention that binds to the cell expressing CD19. In a specific aspect, the present invention provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing CD19, wherein the methods comprise contacting the population of cancer cells expressing CD19 with an anti-CD19 CART cell of the invention that binds to the cell expressing CD19. In one aspect, the present invention provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing CD19, wherein the methods comprise contacting the population of cancer cells expressing CD19 with an anti-CD19 CART cell of the invention that binds to the cell expressing CD19.In certain aspects, the anti-CD19 CART cell of the invention reduces the quantity, number, amount, or percentage of cells and / or cancerous cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a carrier individual, or animal model, of myeloid leukemia or other cancer associated with CD19-expressing cells, relative to a negative control. In one aspect, the individual is a human being.

[00318] The present invention also provides methods for preventing, treating and / or controlling a disease associated with cells expressing CD19 (for example, a hematologic cancer or atypical cancer expressing CD19), wherein the methods comprise administering to an individual in need an anti-CD19 CART cell from Petition 870260065699, dated 03 / 07 / 2026, page 158 / 532 151 / 250 invention that binds to the cell expressing CD19. In one aspect, the individual is a human being. Non-limiting examples of disorders associated with cells expressing CD19 include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma), and cancers (such as hematologic cancers or atypical cancers expressing CD19).

[00319] The present invention also provides methods for preventing, treating and / or controlling a disease associated with cells expressing CD19, wherein the methods comprise administering to an individual in need an anti-CD19 CART cell of the invention that binds to the cell expressing CD19l. In one aspect, the individual is a human being.

[00320] The present invention provides methods for preventing cancer recurrence associated with CD19-expressing cells, wherein the methods comprise administering to an individual in need of such treatment an anti-CD19 CART cell of the invention that binds to the CD19-expressing cell. In one aspect, the methods comprise administering to the individual in need of such treatment an effective amount of an anti-CD19 CART cell described herein that binds to the CD19-expressing cell in combination with an effective amount of another therapy. Combined therapies

[00321] A CAR-expressing cell described herein may be used in combination with other known agents and therapies. Administered in combination, in this descriptive report, means that two (or more) different treatments are provided to the individual during the individual's course of the disorder, for example, the two or more treatments are provided after the individual has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has been discontinued. Petition 870260065699, dated 03 / 07 / 2026, page 159 / 532 152 / 250 for other reasons. In some modalities, the provision of one treatment still occurs when the provision of the second treatment begins, so there is overlap in terms of administration. Sometimes, in this descriptive report, this is referred to as simultaneous or concomitant provision. In other modalities, the provision of one treatment ends before the provision of the other treatment begins. In some modalities, in either case, the treatment is more effective because of the combined administration. For example, the second treatment is more effective, for example, an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if it had been administered in the absence of the first treatment, or the analogous situation is seen with the first treatment.In some modalities, the provision is such that the reduction in a symptom or other parameter related to the disorder is greater than would be observed with one treatment provided in the absence of the other. The effect of the two treatments may be partially additive, entirely additive, or superior to additive. The provision may be such that an effect of the first treatment provided is still detectable when the second is provided.

[00322] A CAR-expressing cell described herein and at least one additional therapeutic agent may be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the CAR-expressing cell described herein may be administered first and the additional agent may be administered second, or the order of administration may be reversed.

[00323] In additional aspects, a CAR-expressing cell described here can be used in a combined treatment regimen with surgery, chemotherapy, radiation, immunosuppressive agents, Petition 870260065699, dated 03 / 07 / 2026, page 160 / 532 153 / 250 such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, Cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation and peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971.

[00324] In one embodiment, a CAR-expressing cell described herein may be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)).an alkaloid of vinca (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an antibody against immune cells (e.g., alemtuzumab, gentuzumab, rituximab, tositumomab), an antimetabolite (including, for example, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a glucocorticoid-induced TNFR-related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin, or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide).

[00325] General chemotherapeutic agents considered for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), injectable busulfan (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), liposomal cytarabine. Petition 870260065699, dated 03 / 07 / 2026, page 161 / 532 154 / 250 injectable (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), liposomal daunorubicin citrate injectable (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium 90 / MX-DTPA), pentostatin, polyfeprosan 20 implant with carmustine (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®),vimblastine (Velban®), vincristine (Oncovin®) and vinorelbine (Navelbine®).,

[00326] Examples of alquiling agents include, among others, nitrogen mustards, derivatives of etilenimine, alquil sulfonates, nitrosoureias and triazenos: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, RevimmuneTM), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylnomelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), estreptozocin (Zanosar®), and dacarbazine (DTIC-Dome®). Petition 870260065699, 03 / 07 / 2026, pág. 162 / 532 155 / 250 Additional exemplary alkylating agents include, but are not limited to, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysin and phenylalanine mustard, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and mechlorethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®);Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and Bendamustine HCl (Treanda®).

[00327] Exemplary mTOR inhibitors include, for example, temsirolimus; ridaforolimo (formerly known as deferolimo, (1R,2R,4S)-4-[(2R)2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03 / 064383); everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-Bis[(3S)-3-methylmorpholin-4 Petition 870260065699, dated 03 / 07 / 2026, p. 163 / 532 156 / 250 yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-Amino8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morolinium-4yl]methoxy]butyl]-L-arginylglycyl-La-aspartyl-L-serine-, internal salt (SF1126, CAS 936487-67-1) and XL765.

[00328] Exemplary immunomodulators include, for example, afutuzumab (provided by Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2, and interferon γ, CAS 951209-71-5, provided by IRX Therapeutics).

[00329] Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin and rubidomycin hydrochloride, Cerubidine®); liposomal daunorubicin (liposomal daunorubicin citrate, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin.

[00330] Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®) and vindesine (Eldisine®); vinblastine (also known as vinblastine sulfate, vincaleucoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

[00331] Exemplary proteasome inhibitors include bortezomib (Velcade®); carfilzomib (PX-171-007, (S)-4-Methyl-N-((S)1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN) Petition 870260065699, 03 / 07 / 2026, page 164 / 532 157 / 250 9708); delanzomib (CEP-18770); and O -Methyl-N-[(2-methyl-5thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[00332] Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as, for example, a GITR fusion protein described in U.S. Patent No. 6,111,090, Patent European Patent No. 090505B1, US Patent No. 8,586,023, PCT Publication Nos. WO 2010 / 003118 and 2011 / 090754, or an anti-GITR antibody described, for example, in US Patent No. 7,025,962, European Patent No. 1947183B1, US Patent No. 7,812,135, US Patent No. 8,388,967, US Patent No. 8,591,886, European Patent No. EP 1866339, PCT Publication No. WO 2011 / 028683, PCT Publication No. WO 2013 / 039954, PCT Publication No. WO2005 / 007190, PCT Publication No. WO 2007 / 133822, PCT Publication No. WO2005 / 055808, PCT Publication No. WO 99 / 40196, PCT Publication No. WO 2001 / 03720, PCT Publication No. WO99 / 20758, PCT Publication No. WO2006 / 083289, PCT Publication No. WO 2005 / 115451, US Patent No. 7,618,632, PCT Publication No. WO 2011 / 051726.

[00333] In one embodiment, a CAR-expressing cell described herein is administered to an individual in combination with an mTOR inhibitor, for example, a rapalog such as everolimus. In one embodiment, the mTOR inhibitor is administered before the CAR-expressing cell. For example, in one embodiment, the mTOR inhibitor may be administered before apheresis of the cells. In one embodiment, the individual has CLL.

[00334] In one embodiment, a CAR-expressing cell described herein is administered to an individual in combination with a GITR agonist, for example, a GITR agonist described herein. In Petition 870260065699, dated 03 / 07 / 2026, page 165 / 532 In one modality, the GITR agonist is administered before the CAR-expressing cell. For example, in one modality, the GITR agonist may be administered before apheresis of the cells. In another modality, the individual has CLL.

[00335] Drugs that inhibit calcium-dependent calcineurin phosphatase (cyclosporines and FK506) or that inhibit p70S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. In a further aspect, the cell compositions of the present invention can be administered to a patient in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, ablative T-cell therapy using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, and / or antibodies such as OKT3 or CAMPATH. In one aspect, the cell compositions of the present invention are administered after ablative B-cell therapy, such as agents that react with CD20, for example, Rituxan.For example, in one embodiment, individuals may undergo standard treatment with high-dose chemotherapy, followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, individuals receive an infusion of expanded immune cells of the present invention. In a further embodiment, expanded cells are administered before or after surgery.

[00336] In one embodiment, the individual may receive an agent that reduces or improves a side effect associated with the administration of a CAR-expressing cell. Side effects associated with the administration of a CAR-expressing cell include, among others, CRS and hemophagocytic lymphohistiocytosis (HLH). Petition 870260065699, dated 03 / 07 / 2026, page 166 / 532 159 / 250, also called macrophage activation syndrome (MAS). Symptoms of CRS include high fever, nausea, transient hypotension, hypoxia, and the like. Thus, the methods described herein may involve administering a CAR-expressing cell described herein to an individual and further administering an agent to control elevated levels of a soluble factor resulting from treatment with a CAR-expressing cell. In one embodiment, the elevated soluble factor in the individual is one or more of IFN-γ, TNFα, IL-2, and IL-6. Therefore, an agent administered to treat this side effect may be an agent that neutralizes one or more of these soluble factors. Such agents include, among others, a steroid, a TNFα inhibitor, and an IL-6 inhibitor. An example of a TNFα inhibitor is entanercept. An example of an IL-6 inhibitor is tocilizumab (toc).

[00337] In one modality, the individual may receive an agent that enhances the activity of a CAR-expressing cell. For example, in one modality, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, for example, programmed cell death protein 1 (PD1), may, in some modalities, decrease the ability of a CAR-expressing cell to mount an effector immune response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. Inhibition of an inhibitory molecule, for example, by inhibition at the DNA, RNA, or protein level, may optimize the performance of a CAR-expressing cell. In some modalities, an inhibitory nucleic acid, for example, a dsRNA, siRNA, or shRNA, may be used to inhibit the expression of an inhibitory molecule in the CAR-expressing cell. In one embodiment, the inhibitor is an shRNA.In one embodiment, the inhibitory molecule is inhibited within a cell expressing CAR. In these embodiments, a dsRNA molecule, which inhibits the expression of the inhibitory molecule, is... Petition 870260065699, dated 03 / 07 / 2026, page 167 / 532 160 / 250 linked to the nucleic acid that encodes a component, for example, all components, of the CAR. In one embodiment, the inhibitor of an inhibitory signal may be, for example, an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent may be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2, or CTLA4 (e.g., ipilimumab (also designated as MDX-010 and MDX-101 and marketed as Yervoy®; BristolMyers Squibb; Tremelimumab (IgG2 monoclonal antibody made available by Pfizer, formerly known as ticilimumab, CP-675,206)). In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In another embodiment, the agent is an antibody or antibody fragment that binds to LAG3.

[00338] PD1 is an inhibitory member of the CD28 family of receptors which also includes CD28, CTLA-4, ICOS, and BTLA.(Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to negatively regulate T cell activation when bound to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1. Antibodies, antibody fragments, and other inhibitors of PD1, PD-L1, and PD-L2 are available in the art and can be used in combination with a CD19 CAR described herein. For example, nivolumab (also designated as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that bind. Petition 870260065699, dated 03 / 07 / 2026, p. 168 / 532 161 / 250 specifically PD1 are described in US Patent 8,008,449 and WO2006 / 121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD1 monoclonal antibodies are described in WO2009 / 101611. Lambrolizumab (also designated as MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD1. Lambrolizumab and other humanized anti-PD1 antibodies are described in US Patent 8,354,509 and WO2009 / 114335. MDPL3280A (Genentech / Roche) is a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are described in U.S. Patent No. 7,943,743 and U.S. Publication No. 20120039906. Other anti-PD-L1 binding agents include YW243.55.S70 (variable regions of the light and heavy chains are shown in SEQ IDs 20 and 21 in WO2010 / 077634) and MDX-1 105 (also designated as BMS936559, and, for example, anti-PD-L1 binding agents described in WO2007 / 005874). AMP-224 (B7-DCIg; Amplimmune; for example, described in WO2010 / 027827 and WO2011 / 066342), is a soluble Fc fusion receptor of PD-L2 that blocks the interaction between PD1 and B7-H1. Other anti-PD1 antibodies include AMP 514 (Amplimmune), among others, for example, the anti-PD1 antibodies described in US Patent 8,609,089, US 2010028330 and / or US 20120114649.

[00339] In some embodiments, the agent that enhances the activity of a cell expressing CAR may be, for example, a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or a fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, for example, a polypeptide comprising an intracellular signaling domain as described herein. In some embodiments, the polypeptide that is associated with a positive signal Petition 870260065699, dated 03 / 07 / 2026, page 169 / 532 162 / 250 may include a co-stimulatory domain of CD28, CD27, ICOS, for example, an intracellular signaling domain of CD28, CD27 and / or ICOS, and / or a primary signaling domain, for example, of CD3 zeta, as described herein. In one embodiment, the fusion protein is expressed by the same cell that expressed the CAR. In another embodiment, the fusion protein is expressed by a cell, for example, a T cell that does not express an anti-CD19 CAR.

[00340] In one embodiment, the agent that enhances the activity of a CAR-expressing cell described here is miR-17-92. Pharmaceutical compositions and treatments

[00341] The pharmaceutical compositions of the present invention may comprise a cell expressing CAR, for example, a plurality of cells expressing CAR, as described herein, in combination with one or more pharmaceutically or physiologically acceptable vehicles, diluents or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate-buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are, in one aspect, formulated for intravenous administration.

[00342] The pharmaceutical compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented). 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 doses can be determined by clinical studies.

[00343] In one embodiment, the pharmaceutical composition is substantially free of, for example, there are no detectable levels of Petition 870260065699, dated 03 / 07 / 2026, page 170 / 532 163 / 250 a contaminant, for example, selected from the group consisting of endotoxin, mycoplasma, replicating competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual coated anti-CD3 / anti-CD28 beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture medium components, vector packaging cell or plasmid components, bacteria and fungi. In one embodiment, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes group A.

[00344] When an immunologically effective amount, an effective antitumor amount, an effective tumor-inhibiting amount, or a therapeutic amount is indicated, the precise amount of the compositions of the present invention to be administered may be determined by a physician taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and patient condition. It may be stated in general that a pharmaceutical composition comprising the T cells described herein may be administered at a dose of 10⁴ to 10⁹ cells / kg of body weight, in some cases 10⁵ to 10⁶ cells / kg of body weight, including all whole numbers within these ranges. The T cell compositions may also be administered multiple times at these doses. The cells may be administered by infusion techniques that are commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[00345] In certain respects, it may be desirable to administer activated T cells to an individual and then subsequently repeat the blood collection (or perform apheresis), activating the T cells present there. Petition 870260065699, dated 03 / 07 / 2026, page 171 / 532 164 / 250 according to the present invention and reinfuse the patient with these activated and expanded T cells. This process can be performed multiple times in a few weeks. In certain aspects, T cells collected from 10 cc to 400 cc of blood can be activated. In certain aspects, T cells collected from 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc or 100 cc of blood are activated.

[00346] The administration of the compositions in question can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient via arterial, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous (iv) injection, or intraperitoneal injection. In one aspect, the T-cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T-cell compositions of the present invention are administered by iv injection. The T-cell compositions can be injected directly into a tumor, lymph node, or site of infection.

[00347] In one exemplary aspect, individuals may undergo leukapheresis, in which leukocytes are collected, enriched, or depleted ex vivo to select and / or isolate cells of interest, for example, T cells. These T cell isolates may be expanded by methods known in the art and treated in such a way that one or more CAR constructs of the invention may be introduced, thus creating a CAR T cell of the invention. Individuals who require it may subsequently undergo standard treatment with high-dose chemotherapy, followed by blood stem cell transplantation. In certain aspects, after or simultaneously with transplantation, individuals receive an infusion of expanded CAR T cells of the present invention. In one aspect Petition 870260065699, dated 03 / 07 / 20...

Claims

CLAIMS 1. Isolated chimeric antigen receptor (CAR) molecule, characterized in that it comprises: (i) an antibody or antibody fragment comprising a humanized anti-CD19 binding domain, (ii) a transmembrane domain and (iii) an intracellular signaling domain, wherein the humanized anti-CD19 binding domain comprises: a light chain complementarity-determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 25, a light chain complementarity-determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 26, a light chain complementarity-determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO: 27, a heavy chain complementarity-determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 19,a heavy chain complementarity-determining region 2 (HC CDR2) comprising the amino acid sequence of any of the SEQ ID NOs: 22, 21 or 23, and a heavy chain complementarity-determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO:

24.

2. Isolated CAR molecule according to claim 1, characterized in that: (i) the anti-CD19 binding domain comprises a variable light chain region and a variable heavy chain region of an amino acid sequence listed in SEQ ID NOs: 32, 31 or 33-42; Petition 870260065699, dated 03 / 07 / 2026, p. 259 / 532 2 / 6 (ii) the anti-CD19 binding domain comprises a variable light chain region listed in SEQ ID NOs: 32, 31 or 33 to 42; (iii) the anti-CD19 binding domain comprises a variable heavy chain region listed in SEQ ID NOs: 32, 31 or 33 to 42; or (iv) the anti-CD19 binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:

12.

3. Isolated CAR molecule, according to claim 1 or 2, characterized in that: (i) the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154; (ii) the transmembrane domain comprises a transmembrane domain of the alpha chain of CD8; or (iii) the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

15.

4. Isolated CAR molecule, according to any one of claims 1 to 3, characterized in that the humanized anti-CD19 binding domain is connected to the transmembrane domain by a linkage region.

5. Isolated CAR molecule according to claim 4, characterized in that: (i) the linkage region is an alpha linkage of CD8; or (ii) the linkage region comprises the amino acid sequence of SEQ ID NO:

14. Petition 870260065699, dated 03 / 07 / 2026, p. 260 / 532 3 / 6 6. Isolated CAR molecule, according to any one of claims 1 to 5, characterized in that the intracellular signaling domain comprises a co-stimulatory domain.

7. Isolated CAR molecule, according to claim 6, characterized in that: (i) the co-stimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18) and 4-1BB (CD137); or (ii) the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:

16.

8. Isolated CAR molecule, according to any one of claims 1 to 7, characterized in that: (i) the intracellular signaling domain comprises a primary signaling domain; (ii) the intracellular signaling domain comprises a 4-1BB functional signaling domain and / or a CD3 zeta functional signaling domain; (iii) the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 16 and / or the amino acid sequence of SEQ ID NO: 17; (iv) the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 16 and / or the amino acid sequence of SEQ ID NO: 43; or (v) the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43, wherein the sequences comprising the intracellular signaling domain are expressed in the same reading frame and as a single polypeptide chain.Petition 870260065699, dated 03 / 07 / 2026, p. 261 / 532 4 / 6.

9. Isolated CAR molecule, according to any one of claims 1 to 8, characterized in that it further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:

13.

10. Humanized anti-CD19 binding domain, characterized in that it comprises: a light chain complementarity-determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 25, a light chain complementarity-determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 26, a light chain complementarity-determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO: 27, a heavy chain complementarity-determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 19, a heavy chain complementarity-determining region 2 (HC CDR2) comprising the amino acid sequence of any of the SEQ ID NOs: 22, 21 or 23, and a heavy chain complementarity-determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO:

24.

11. Humanized anti-CD19 binding domain, according to claim 10, characterized in that it comprises: (i) any variable region of the light chain listed in SEQ ID NOs: 32, 31 or 33 to 42; (ii) any variable region of the heavy chain listed in SEQ ID NOs: 32, 31 or 33 to 42; or Petition 870260065699, dated 03 / 07 / 2026, page 262 / 532 5 / 6 (iii) a variable region of the light chain and a variable region of the heavy chain of an amino acid sequence from any of the SEQ ID NOs: 32, 31 or 33 to 42.

12. Method, characterized in that it comprises: (i) generating a population of RNA-manipulated cells comprising introducing a transcribed RNA or synthetic RNA in vitro into a cell, wherein the RNA comprises a nucleic acid molecule encoding the CAR molecule, as defined in any one of claims 1 to 9; or (ii) producing a transcribed RNA in vitro encoding a CD19 CAR, comprising performing in vitro transcription on a DNA sequence encoding a CD19 CAR, wherein the CD19 CAR is the CAR molecule, as defined in any one of claims 1 to 9.

13. Use of the CAR molecule, as defined in any one of claims 1 to 9, characterized in that it is for the manufacture of a medicament for the treatment of a disease associated with CD19 expression, wherein the disease associated with CD19 expression is selected from the group consisting of cancer or malignancy, precancerous condition and non-cancer-related indication associated with CD19 expression.

14. Use, according to claim 13, characterized in that the disease associated with CD19 expression is a hematologic cancer selected from the group consisting of B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolinocytic leukemia, plasmacytoid dendritic neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, follicular lymphoma. Petition 870260065699, dated 03 / 07 / 2026, p. 263 / 532 6 / 6 small or large, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasma lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia and combinations thereof.

15. Use according to claim 13 or 14, characterized in that the cell is administered in combination with: (i) an agent that increases the effectiveness of the cell; (ii) an agent that improves one or more side effects associated with the administration of the cell; (iii) an agent that treats the disease associated with CD19; (iv) an agent that inhibits an inhibitory molecule chosen from PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160 or 2B4; (v) a PD1 CAR comprising an extracellular PD1 domain, a transmembrane domain and an intracellular signaling domain; (vi) a GITR agonist; (vii) an IL-6 inhibitor; or (viii) an mTOR inhibitor.

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