CD20 therapy, CD22 therapy, and combination therapy with CD19 chimeric antigen receptor (CAR) expressing cells

Abstract

This invention provides a method for treating subjects (e.g., mammals) with diseases associated with CD19 expression. [Solution] The present invention provides cells expressing a CD19-binding CAR molecule, such as CD19CAR, for use in the treatment of subjects having a disease associated with CD19 expression, wherein the subject has received, is receiving, or is scheduled to receive one or more B cell inhibitors, the B cell inhibitor comprising one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. The present invention also provides a composition comprising the cells and a method for treating the disease.

Description

[Technical Field]

[0001] This application claims priority to U.S. Patent Application No. 62 / 144,615, filed April 8, 2015, U.S. Patent Application No. 62 / 144,497, filed April 8, 2015, U.S. Patent Application No. 62 / 144,639, filed April 8, 2015, U.S. Patent Application No. 62 / 207,255, filed August 19, 2015, and U.S. Patent Application No. 62 / 263,423, filed December 4, 2015, the contents of which these applications are incorporated herein by reference in their entirety.

[0002] The present invention generally relates to the use of T cells modified to express a chimeric antigen receptor (CAR) for treating diseases associated with the expression of surface antigen classification 19 protein (CD19), in combination, as appropriate, with one or more B cell inhibitors, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. [Background technology]

[0003] Many patients with B-cell malignancies are untreatable with standard treatments. Furthermore, traditional treatment options often have serious side effects. While various attempts have been made in cancer immunotherapy, achieving clinical efficacy remains a very difficult goal due to numerous obstacles. Hundreds of so-called tumor antigens have been identified, but these are generally autologous and therefore have low immunogenicity. Moreover, tumors use a variety of mechanisms to resist the initiation and propagation of immune attacks.

[0004] Recent advances using chimeric antigen receptor (CAR)-modified autologous T-cell (CART) therapy, which relies on the redirection of T cells to suitable cell surface molecules on cancer cells such as B-cell malignancies, have shown promising results in leveraging the power of the immune system to treat B-cell malignancies and other cancers [see, e.g., Sadelain et al., Cancer Discovery 3:388-398 (2013)]. Clinical outcomes of mouse-derived CART19 (i.e., "CTL019") have demonstrated effectiveness in achieving a complete response in patients with CLL as well as in pediatric ALL [see, e.g., 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 of chimeric antigen receptors on genetically modified T cells to recognize and destroy target cells, the ability to proliferate and persist over long periods to investigate leukemia relapse is necessary for the success of therapeutic T cell therapy. The variability in the quality of T cells due to anergy, suppression, or depletion will affect the performance of CAR-transformed T cells, and at present, there are limitations to their control by those skilled in the art. To be effective, CAR-transformed patient T cells need to sustain and maintain the ability to proliferate in response to allogeneic antigens. It has been demonstrated that T cells from ALL patients can perform this using CART19, which includes mouse scFv [see, for example, Grupp et al., NEJM 368:1509-1518 (2013)]. [Overview of the project] [Means for solving the problem]

[0005] This disclosure features, at least in part, a method for treating disorders related to the expression of surface antigen classification 19 protein (CD19) (e.g., OMIM accession number 107265, Swiss Prot. accession number P15391). In certain embodiments, the disorder is cancer, e.g., hematological cancer. In some embodiments, the method involves administering a chimeric antigen receptor (CAR) molecule that binds to CD19 in combination with a B cell inhibitor, e.g., one or more (e.g., one, two, three or more) B cell inhibitors. In some embodiments, the B cell inhibitors are selected from inhibitors of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, or ROR1, or combinations thereof. In some embodiments, the combination maintains or has better clinical efficacy compared to either treatment alone. In some embodiments, the methods described herein involve using cells modified to express a CD19-binding CAR molecule, such as T cells, in combination with a B cell inhibitor [e.g., an antibody against a second B target, e.g., CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, or ROR1 (e.g., a monospecific or bispecific antibody)], or CAR-expressing cells that bind to a second B cell target, such as CAR-expressing immunoeffector cells, or a combination thereof, to address impairments related to CD19 expression. The disclosure further features novel antigen-binding domains and CAR molecules directed towards CD20 and CD22, and their use, for example, as monotherapy or in combination therapy.

[0006] Accordingly, in one embodiment, the present invention relates to a method for treating subjects (e.g., mammals) having a disease associated with CD19 expression. The method comprises administering a CD19 inhibitor, for example, a CD19-binding CAR molecule as described herein, to the subject in combination with a B cell inhibitor. For example, the method comprises administering to the subject an effective number of cells expressing a CD19-binding CAR molecule, for example, a CD19-binding CAR molecule as described herein (e.g., wild-type or mutant CD19), in combination with a B cell inhibitor. In certain embodiments, the B cell inhibitor is selected from CD10 inhibitors, e.g., one or more CD10 inhibitors described herein; CD20 inhibitors, e.g., one or more CD20 inhibitors described herein; CD22 inhibitors, e.g., one or more CD22 inhibitors described herein; CD34 inhibitors, e.g., one or more CD34 inhibitors described herein; CD123 inhibitors, e.g., one or more CD123 inhibitors described herein; FLT-3 inhibitors, e.g., one or more FLT-3 inhibitors described herein; ROR1 inhibitors, e.g., one or more ROR1 inhibitors described herein; CD79b inhibitors, e.g., one or more CD79b inhibitors described herein; CD179b inhibitors, e.g., one or more CD179b inhibitors described herein; CD79a inhibitors, e.g., one or more CD79a inhibitors described herein; or any combination thereof. In a particular embodiment, a method for treating a subject having B-cell leukemia or B-cell lymphoma is disclosed, comprising administering to the subject an effective number of one or more cells expressing a CD19-binding CAR molecule in combination with one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0007] In a related embodiment, the Disclosure provides a method for reducing the proliferation of CD19-expressing cells, for example, by administering a combination therapy as described herein to a subject, for example, a patient in need of such treatment, for example, by administering a CD19 inhibitor in combination with a B cell inhibitor, for example, one or more B cell inhibitors as described herein. In another embodiment, the Disclosure provides a method for selectively killing CD19-expressing cells, for example, by administering a combination therapy as described herein to a subject, for example, a patient in need of such treatment, for example, by administering a CD19 inhibitor in combination with a B cell inhibitor, for example, one or more B cell inhibitors as described herein. In a particular embodiment, the Disclosure provides a method for providing antitumor immunity in a subject, for example, a mammal, comprising administering an effective amount of a combination (e.g., one or more CAR-expressing cells) as described herein to the mammal.

[0008] In one embodiment, the Disclosure provides a method for preventing CD19-negative relapse in a mammal, comprising administering one or more B-cell inhibitors to the mammal, wherein the B-cell inhibitors include one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0009] In another aspect, the disclosure provides a method for treating subjects having a disease associated with CD19 expression, such as DLBCL (e.g., primary DLBCL). The method comprises administering to the subject one or more effective numbers of cells expressing a CD19-binding CAR molecule, such as CD19 CAR, optionally in combination with a PD1 inhibitor. The subject may have, or be identified as having, at least 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% CD3+ / PD1+ cancer cells, such as DLBCL cells.

[0010] In some embodiments, the disclosure provides a method for treating subjects having a disease associated with CD19 expression, such as DLBCL. The method comprises administering to the subject an effective number of one or more cells expressing a CD19-binding CAR molecule, such as a CD19 CAR, in combination with a PD-L1 inhibitor. The subject may have, or be identified as having, cells in the cancer, e.g., cancer microenvironment, that are double positive for CD19 and PD-L1, such as 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1%.

[0011] In one embodiment, the Disclosure provides one or more B cell inhibitors for use in the treatment of subjects having a disease related to CD19 expression, comprising one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, wherein the subject has received, is receiving, or is scheduled to receive cells expressing a CD19-binding CAR molecule, such as a CD19 CAR.

[0012] Timing and dosage of concomitant administration One or more of the treatments described herein can be administered to a subject substantially simultaneously or in any order. For example, CD19 inhibitors, e.g., CD19 CAR-expressing cells as described herein, one or more B-cell inhibitors, and / or, as appropriate, at least one additional therapeutic agent may be administered simultaneously or sequentially in the same or different compositions.

[0013] In the case of sequential administration, CAR-expressing cells described herein (e.g., CD19 CAR-expressing cells, CD20 CAR-expressing cells, or CD22 CAR-expressing cells) may be administered first, followed by the additional drug, or the order of administration may be reversed. In some embodiments, the first treatment (e.g., CAR-expressing cells, e.g., CD19 CART cells, CD20 CART cells, or CD22 CART cells) continues when the second treatment is introduced, while in other embodiments, the first treatment is discontinued before, after, or simultaneously with the introduction of the second treatment. In the case of sequential administration, in some embodiments, the second treatment is initiated after a predetermined time or after the subject has shown one or more signs of relapse, or is likely to relapse. Signs may include, for example, the presence of cancer cells with impaired targets of the first treatment, e.g., CD19, CD20, or CD22. The impairment may include, for example, frameshift mutations and / or immature stop codons.

[0014] In other embodiments, two or more treatments (e.g., CD19 CAR-expressing cells and B-cell inhibitors) are administered simultaneously. While we do not wish to be bound by theory, in some embodiments, the simultaneous administration of treatments may reduce the likelihood of relapse and / or delay relapse.

[0015] When administered in combination, the first treatment (e.g., CAR therapy, e.g., CAR-expressing cells directed towards CD19, CD20, or CD22) and the additional agent (e.g., a second or third agent, e.g., a B-cell inhibitor), or all of them, may be administered in a higher or higher dose than, or lower or equal to, the amount or dosage of each agent used individually, for example, as monotherapy. In a particular embodiment, the amount or dosage of the first treatment, the second treatment, optionally the third treatment, or all of them administered is lower or lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, for example, as monotherapy. In other embodiments, the amount or dosage of the first, second, and optionally third treatments, or all of them, that result in the desired effect (e.g., treatment of cancer) is lower or less than the amount or dosage of each drug used individually, for example, as monotherapy, that would be required to achieve the same therapeutic effect (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower or less). In certain embodiments, the lower dose results in a reduction of side effects compared to those seen when the usual dose (the monotherapy dose) is administered.

[0016] In one embodiment, the treatment comprises a population of cells. In one embodiment, the cells are immune effector cells, for example, CAR-expressing cells.

[0017] Alternatively, or in combination with the methods described herein, a method is disclosed that includes a diagnostic step or a patient selection step, for example, a step as described below.

[0018] In one embodiment, the present invention provides a method for evaluating subjects, such as patients, for relapse status (e.g., relapse or non-relapse after CAR therapy). In one embodiment, the method identifies subjects, such as patients ("relapsed cases"), or subjects, such as patients, who are likely to relapse, or subjects, such as patients, who have not relapsed, or subjects, such as patients ("non-relapsed cases"), or subjects, such as patients, who are unlikely to relapse, after treatment with CAR therapy (e.g., CD19 CART therapy, e.g., CD19 CART therapy as described herein, e.g., CTL019 therapy). In one embodiment, relapse status (e.g., relapse or non-relapse after CART therapy) is determined by assaying one or more features of CD19.

[0019] In one embodiment, one or more features of CD19 include mutations, such as changes in the nucleic acid sequence (e.g., insertions, deletions, substitutions, or combinations thereof), changes at the nucleic acid level, changes in the protein sequence, or changes at the protein level, or combinations thereof. In one embodiment, a recurrent example has one or more mutations in CD19, for example, one or more mutations (e.g., insertions or deletions) in exon 2 of CD19. In another embodiment, a recurrent example has one or more mutations in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of CD19. In another embodiment, the mutation generates an immature stop codon, for example, in exon 2 of CD19, for example, by an insertion or deletion leading to a frameshift. In another embodiment, the mutation is one of the mutations in Table 31.

[0020] In one embodiment, a CD19 feature is compared to a reference feature. For example, if the feature is a sequence (e.g., a protein or nucleic acid sequence from a biological sample), the reference feature may be the wild-type sequence of CD19 (e.g., a protein or nucleic acid sequence). The feature may also be the percentage of cells in the sample that have the mutant sequence. If the feature is a level (e.g., a protein or nucleic acid level), the reference feature may be the wild-type level of CD19 (e.g., a protein or nucleic acid level). The feature may also be the level of protein or nucleic acid in the sample. The feature may also be the percentage of cells in the sample that have a level of protein or nucleic acid above a given threshold.

[0021] In one embodiment, a method is provided for identifying subjects having cancer, such as hematological cancers, such as CLL or ALL, as recurrent or non-recurrent after treatment including CAR therapy, such as CD19 CART therapy. The method includes (1) obtaining a sample from the subject (e.g., an apheresis sample obtained from the subject's blood; and / or, for example, a manufactured product sample, such as genetically modified T cells obtained from the subject's blood); (2) determining a CD19 feature, such as sequence or level, as described herein; and (3) comparing the determined CD19 feature with a reference feature (as appropriate) (in this case, the difference between the determined feature and the reference feature, e.g., a statistically significant difference, indicates recurrence to CAR therapy); and (4) identifying the subject as recurrent or non-recurrent to CAR therapy, for example, based on the determined CD19 feature. In one embodiment, the presence or absence of a CD19 feature is the presence or absence of an immature stop codon, for example, due to an insertion or deletion leading to a frameshift. In one embodiment, the presence of a CD19 feature is a mutation as shown in Table 31.

[0022] In one embodiment, the method provided comprises (1) obtaining a sample from a subject (e.g., an apheresis sample obtained from the subject's blood; and / or, for example, a manufactured product sample, e.g., genetically modified T cells obtained from the subject's blood, e.g., a manufactured CART19 product); (2) determining a CD19 feature, e.g., sequence or level, as described herein; and (3) comparing the determined CD19 feature with a reference feature (as appropriate), wherein the presence of the CD19 feature (e.g., a difference between the determined feature and the reference feature, e.g., a statistically significant difference) foreshadows relapse in response to CAR therapy. In one embodiment, the presence of the CD19 feature is the presence of an immature stop codon, e.g., an insertion or deletion leading to a frameshift. In another embodiment, the presence of the CD19 feature is a mutation as shown in Table 31.

[0023] In one embodiment, a method is provided for determining recurrence in subjects with cancer, such as hematological malignancies like CLL or ALL, after treatment including CAR therapy, such as CD19 CAR therapy as described herein. The method includes determining the CD19 feature in a sample obtained before recurrence. In one embodiment, the presence of the CD19 feature (e.g., a difference between the determined feature and a reference feature, e.g., a statistically significant difference) indicates recurrence after CAR therapy. In one embodiment, the presence of the CD19 feature is the presence of an immature stop codon, such as an insertion or deletion leading to a frameshift. In another embodiment, the presence of the CD19 feature is a mutation as shown in Table 31.

[0024] In one embodiment, a method is provided for evaluating subjects having cancer, such as hematological cancers such as CLL or ALL. The method includes evaluating the subjects by obtaining a value for the subject's recurrence status, which includes one or more characteristics of CD19, for example, one or more measures of CD19 characteristics as described herein.

[0025] In one embodiment, a method is provided for evaluating or monitoring the effectiveness of CAR therapy, such as CD19 CART therapy, in a subject having cancer, the method comprising obtaining a value for the subject's recurrence status, which includes one or more characteristics of CD19, such as one or more measures of the characteristics of CD19 as described herein.

[0026] In one embodiment, a method is provided to provide a prediction of the success rate of CAR therapy in a subject having cancer, for example, CD19 CART therapy, for example, CD19 CART therapy as described herein, comprising the steps of: preparing a biological sample from the subject; determining one or more characteristics of CD19, for example, one or more characteristics of CD19 as described herein; and providing a prognosis to the subject based on the determined characteristics.

[0027] In some embodiments, the Disclosure provides, for example, a method or assay for identifying a subject having cancer that has an increased or decreased likelihood of responding to a treatment including chimeric antigen receptor (CAR) therapy, the method being (1) Obtain a sample from the subject; (2)(i) Levels of one or more markers listed in Table 29 in the sample, (ii) CD19 characteristics, for example, mutations, for example, mutations that cause frameshifts or immature stop codons or both, (iii)T REG At the cellular level or activity level To determine the value for one or more of them; (3) Comparing the determined values, e.g., (i), (ii), or (iii) or combinations thereof, to reference values ​​(as appropriate) (in this case, the difference between the determined value and the reference value, e.g., a statistically significant difference, foreshadows the subject's response to CAR therapy); and (4) Identifying the subjects based on the determined values ​​as cases with a complete response, partial response, or no response to CAR therapy, or as cases with relapse or no relapse. Includes.

[0028] In a particular embodiment, any of the methods described above may further encompass the following: (i) the level or activity of one or more markers listed in Table 29, (ii) characteristics of CD19, e.g., mutations that cause frameshift or immature stop codons or both, or (iii) T in the biological sample REG If no difference is detected with respect to one or more (all) values ​​at the cellular level, for example, if no statistically significant difference is detected, administer a therapeutically effective dose of CAR therapy, such as a therapy involving CD19-expressing cells; (ii) (i) levels or activity of one or more markers listed in Table 29, (ii) characteristics of CD19, e.g., mutations that cause frameshift or immature stop codons or both, or (iii) T in the biological sample REG If a difference is detected with respect to one or more (all) values ​​at the cellular level, for example, a statistically significant difference, administer a therapeutically effective dose of CAR therapy, such as therapy involving CD19-expressing cells, and one or more B-cell inhibitors (e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1 as described herein); or (iii) (i) levels or activity of one or more markers listed in Table 29, (ii) characteristics of CD19, e.g., mutations, e.g., mutations causing frameshift or immature stop codon or both, or (iii) T in the biological sample REGIf a difference is detected with respect to one, two or more (or all) values ​​at the cellular level, for example, a statistically significant difference, discontinue the first treatment, e.g., the treatment involving CD19-expressing cells, and administer a second treatment, e.g., one or more B-cell inhibitors (e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1 as described herein).

[0029] The administration steps (i) to (iii) may be performed before or after the patient evaluation step, as described in the exemplary embodiments below.

[0030] In a particular embodiment, a method for treating a subject having cancer is disclosed. The method is (a) The subject is, (i) Levels of one or more markers listed in Table 29, (ii) CD19 characteristics, for example, mutations that cause frameshift or immature stop codons or both, (iii) T in biological samples REG Cellular level or activity If it has one or more (all) values, obtaining them, for example, by determining them, (b) Depending on the value, the following further include: (i) the level or activity of one or more markers listed in Table 29, (ii) characteristics of CD19, e.g., mutations that cause frameshift or immature stop codons or both, or (iii) T in the biological sample REG If no difference is detected at the cellular level, with respect to one, two or more (or all) of these, for example, if no statistically significant difference is detected, administer a therapeutically effective dose of CAR therapy, such as a therapy involving CD19-expressing cells; (ii) (i) levels or activity of one or more markers listed in Table 29, (ii) characteristics of CD19, e.g., mutations that cause frameshift or immature stop codons or both, or (iii) T in the biological sampleREG If a difference, e.g., a statistically significant difference, is detected for one, two or more (or all) of the levels of cells, then administer to the subject a therapeutically effective dose of a CAR therapy, e.g., a therapy that includes CD19-expressing cells, and one or more B cell inhibitors (e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3 or ROR1 as described herein); or (iii) (i) the level or activity of one or more markers listed in Table 29, (ii) a characteristic of CD19, e.g., a mutation, e.g., a mutation that causes a frameshift or premature stop codon or both, or (iii) T in a biological sample REG If a difference, e.g., a statistically significant difference, is detected for one or more of the levels of cells, then discontinue the first therapy, e.g., a therapy that includes CD19-expressing cells, and administer a second therapy, e.g., one or more B cell inhibitors (e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3 or ROR1 as described herein).

[0031] In another aspect, provided is a method of treating a subject having cancer. The method includes: (a) administering to the subject a therapeutically effective dose of a CAR therapy, e.g., a therapy that includes CD19-expressing cells, (b) (I) obtaining values for (e.g., determining if the subject has) the level of one or more markers listed in Table 29, (II) a characteristic of CD19, e.g., a mutation, e.g., a mutation that causes a frameshift or premature stop codon or both, or (III) T in a biological sample REG level or activity of cells, for one, two or more (all) thereof, and (c) performing one or more of the following depending on the values or determinations in step (b)(I-III): (i) (I) Levels or activity of one or more markers listed in Table 29, (II) Characteristics of CD19, e.g., mutations, e.g., mutations causing frameshift or immature stop codon or both, or (III) T in the biological sample REG If no difference is detected with respect to one or more cellular levels, for example, if no statistically significant difference is detected, administer a therapeutically effective dose of CAR therapy, such as a therapy involving CD19-expressing cells; (ii) (I) Levels or activity of one or more markers listed in Table 29, (II) Characteristics of CD19, e.g., mutations, e.g., mutations causing frameshift or immature stop codon or both, or (III) T in the biological sample REG If a difference is detected with respect to one or more cellular levels, for example, a statistically significant difference, administer a therapeutically effective dose of CAR therapy, such as a therapy involving CD19-expressing cells, and one or more B-cell inhibitors (e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1 as described herein); or (iii) (i) levels or activity of one or more markers listed in Table 29, (ii) characteristics of CD19, e.g., mutations, e.g., mutations causing frameshift or immature stop codon or both, or (iii) T in the biological sample REG If a difference is detected with respect to one or more levels of cells, for example, a statistically significant difference, discontinue the first treatment, for example, the treatment involving CD19-expressing cells, and administer a second treatment, for example, one or more B-cell inhibitors (e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1 as described herein).

[0032] In some embodiments of any of the methods described above, the sample is a biological sample selected from blood, plasma, or serum samples. In certain embodiments, the biological sample is a blood sample. In one embodiment, the sample is an apheresis sample, e.g., T cells obtained from the blood of the subject. In some embodiments, the sample is a manufactured product sample, e.g., genetically modified T cells obtained from the blood of the subject, e.g., a manufactured CAR product, e.g., a manufactured CART19 product.

[0033] In some embodiments, the methods described herein can be used to determine whether a patient is likely to respond to CAR therapy (e.g., CD19 CAR therapy), for example, whether a patient who has never received CAR therapy is likely to respond to CAR therapy, or whether a patient who has received CAR therapy is likely to respond to continued CAR therapy. Generally, the same CD19 features that foreshadow relapses foreshadow a patient's low likelihood of responding to CD19 CAR therapy. Patients identified as having a low likelihood of responding to CD19 CAR therapy may be administered different types of treatment, such as B-cell inhibitors (e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3 or ROR1, CD79b, CD179b or CD179a, as described herein).

[0034] In another embodiment, a method is provided for treating a subject having cancer, for example, a hematological malignancy. In one embodiment, the method comprises determining whether the subject has a difference in CD19 features, for example, a statistically significant difference, compared to a reference feature, and if there is a difference, for example, a statistically significant difference, between the determined feature and the reference feature, treating the subject by administering a therapeutically effective dose of CAR therapy, for example, CART, to the subject. In one embodiment, the feature is a CD19 sequence, for example, a protein or nucleic acid sequence. In one embodiment, the method comprises assaying for the presence or absence of frameshifted CD19, for example, CD19 containing immature stop codons.

[0035] In embodiments of any of the methods described above, the treatment includes administering CD19 CAR-expressing cells in combination with, as appropriate, one or more B-cell inhibitors. In some embodiments, the CD19 CAR therapy is administered concurrently with one or more B-cell inhibitors (e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD179a, as described herein). In some embodiments, the CD19 CAR therapy is administered before one or more B-cell inhibitors. In some embodiments, the CD19 CAR therapy is administered after one or more B-cell inhibitors.

[0036] In some embodiments, if there is a difference between the determined feature and the reference feature, the method includes modifying the CAR product before injection into the subject. In some embodiments, if there is a difference between the determined feature and the reference feature, the method includes modifying the production of the CAR product before injection into the subject. In some embodiments, if there is a difference between the determined feature and the reference feature, the method includes adjusting the CAR injection dose to achieve an anticancer effect.

[0037] In one embodiment, the treatment method includes determining whether the subject has an increased likelihood of responding to CAR therapy, such as CD19 CART therapy, as described herein, by comparing the features of CD19 in a sample from the subject to a reference feature (in this case, the difference in features to the reference feature indicates an increased likelihood of response), and treating the subject by administering a therapeutically effective dose of CAR therapy to the subject.

[0038] In one embodiment, the treatment method includes obtaining a sample from a subject, determining the CD19 features (e.g., frameshift or presence or absence of immature stop codons) compared to a reference feature, and administering a therapeutically effective dose of CAR-expressing cells if the subject is identified as having a statistically significant difference between the CD19 features of the sample and the reference feature in the sample.

[0039] CD19 characteristics can be used to design patient treatment. For example, in one embodiment, if the patient sample contains wild-type CD19, the patient is administered a CD19 inhibitor, e.g., CD19 CAR-expressing cells, e.g., CD19 CART. In one embodiment, if the patient sample contains mutant CD19, e.g., frameshifted CD19, e.g., CD19 containing immature stop codons, the patient is administered a treatment other than a CD19 inhibitor, e.g., the patient is administered another B-cell inhibitor. In one embodiment, if the patient sample contains at least normal levels of CD19, the patient is administered a CD19 inhibitor, e.g., CD19 CAR-expressing cells, e.g., CD19 CART. In one embodiment, if a patient sample contains CD19 below normal levels, the patient is administered a treatment other than a CD19 inhibitor, for example, the patient is administered another B cell inhibitor (e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a as described herein).

[0040] In one embodiment, the treatment method involves obtaining a value for the relapse status of the subject, which includes a measure of the CD19 feature, and, depending on the determination of the relapse status, one, two, three, four or more (three, four, or This includes: (1) identifying subjects as relapsed or non-relapsed; (2) administering CAR therapy; (3) selecting or modifying the administration of CAR therapy; (4) selecting or modifying the schedule or time course of CAR therapy; (5) administering additional drugs in combination with CAR therapy to, for example, relapsed subjects, e.g., one or more B cell inhibitors or checkpoint inhibitors, e.g., checkpoint inhibitors as described herein, or kinase inhibitors, e.g., kinase inhibitors as described herein; (6) administering treatment to relapsed subjects to increase the number of naive T cells in the subject before treatment with CAR therapy; modifying the manufacturing process of CAR therapy, e.g., enriching naive T cells in, for example, subjects identified as relapsed before introducing the nucleic acid encoding the CAR; or (7) selecting an alternative treatment for, for example, relapsed subjects, e.g., standard treatment for a particular cancer (e.g., as described herein); and thereby treating the cancer of the subject.

[0041] In some embodiments, the method includes administering one, two, three or more B-cell inhibitors (e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1 as described herein). For example, in one embodiment, the method includes administering a CD19 inhibitor, e.g., cells expressing a CD19 CAR, in combination with a C10 inhibitor, or in combination with a C10 inhibitor and any combination of an inhibitor of CD20, CD22, CD34, CD123, FLT-3, or ROR1 as described herein. In another embodiment, the method includes administering a CD19 inhibitor, e.g., cells expressing a CD19 CAR, in combination with a C20 inhibitor, or in combination with a C20 inhibitor and any combination of an inhibitor of CD10, CD22, CD34, CD123, FLT-3, or ROR1 as described herein. In another embodiment, the method includes administering a CD19 inhibitor, for example, cells expressing a CD19 CAR, in combination with a C22 inhibitor, or in combination with a C22 inhibitor and any combination of a CD10, CD20, CD34, CD123, FLT-3, or ROR1 inhibitor as described herein. In another embodiment, the method includes administering a CD19 inhibitor, for example, cells expressing a CD19 CAR, in combination with a CD34 inhibitor, or in combination with a CD4 inhibitor and any combination of a CD10, CD20, CD22, CD123, FLT-3, or ROR1 inhibitor as described herein. In another embodiment, the method includes administering a CD19 inhibitor, for example, cells expressing a CD19 CAR, in combination with a CD123 inhibitor, or in combination with a CD123 inhibitor and any combination of a CD10, CD20, CD34, CD22, FLT-3, or ROR1 inhibitor as described herein. In another embodiment, the method includes administering a CD19 inhibitor, for example, cells expressing a CD19 CAR, in combination with a FLT-3 inhibitor, or in combination with a FLT-3 inhibitor in any combination of an inhibitor of CD10, CD20, CD34, CD123, or ROR1 as described herein.In another embodiment, the method includes administering a CD19 inhibitor, for example, cells expressing a CD19 CAR, in combination with a ROR1 inhibitor, or in combination with a ROR1 inhibitor and any combination of CD10, CD20, CD34, CD123, or FLT-3 inhibitors as described herein. In some embodiments, the method includes administering one, two, three or more B-cell inhibitors (for example, one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1, CD79b, CD179b, or CD79a as described herein).

[0042] In some embodiments, the treatment methods described herein further include determining the levels of immune checkpoint molecules (e.g., PD-L1, PD1, LAG3, or TIM3) in a patient sample and administering an immune checkpoint inhibitor (e.g., one or more inhibitors of PD-L1, PD1, LAG3, and TIM3) to the patient, or both. For example, the method may include treating the patient with one or more CAR-expressing cells described herein (e.g., B-cell inhibitors, CD20 CAR, or CD19 CAR in combination with CD22 CAR), and determining the levels of immune checkpoint molecules in the patient before or after the treatment. In some embodiments, the method includes administering an immune checkpoint inhibitor to a patient having elevated immune checkpoint molecule levels compared to a reference level, for example, administering a PD-L1 inhibitor in accordance with elevated PD-L1 levels, administering a PD1 inhibitor in accordance with elevated PD1 levels, administering a LAG3 inhibitor in accordance with elevated LAG3 levels, or administering a TIM3 inhibitor in accordance with elevated TIM3 levels. In some embodiments, the method involves administering an immune checkpoint inhibitor to a patient who has, is, or is scheduled to be treated with one or more CAR-expressing cells as described herein (e.g., CD19 CAR, CD20 CAR, or CD22 CAR in combination with a B-cell inhibitor) and who has, or is identified as having, elevated immune checkpoint molecular levels compared to a reference level.

[0043] composition In some embodiments, the Disclosure provides compositions comprising, for example,: (i) a CD19-binding CAR molecule, e.g., one or more cells expressing a CD19 CAR as described herein, e.g., one or more B cell inhibitors, e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1. In embodiments, (i) and (ii) are provided separately, and in embodiments, (i) and (ii) are mixed.

[0044] In some embodiments, the disclosure provides nucleic acids encoding, for example, (i) a CD19-binding CAR molecule, e.g., a CD19-binding CAR molecule as described herein, e.g., CD19 CAR, and (ii) one or more B cell inhibitors, e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1. In some embodiments, the disclosure provides nucleic acids encoding, for example, (i) a CD19-binding CAR molecule, e.g., a CD19-binding CAR molecule as described herein, e.g., CD19 CAR, and (ii) a CAR molecule that binds to one or more B cell antigens, e.g., CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1. In embodiments, the nucleic acid includes RNA or DNA.

[0045] In some embodiments, the Disclosure provides nucleic acids encoding, for example: (i) a CD19-binding CAR molecule, e.g., a CD19-binding CAR molecule as described herein, e.g., CD19 CAR; and (ii) a CAR molecule binding to one or more B cell antigens, e.g., CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. In embodiments, the nucleic acid comprises RNA or DNA. In embodiments, the nucleic acid sequences encoding (i) and (ii) are located in the same configuration, and, for example, the transcription of the nucleic acid sequences encoding (i) and (ii) proceeds in the same direction. In embodiments, the nucleic acid sequences encoding (i) and (ii) are located in different configurations. In embodiments, a single promoter controls the expression of the nucleic acid sequences encoding (i) and (ii). In embodiments, a nucleic acid encoding a protease cleavage site (e.g., a T2A, P2A, E2A, or F2A cleavage site) is located between the nucleic acid sequences encoding (i) and (ii). In embodiments, the protease cleavage site is positioned so that a cell can express a fusion protein comprising (i) and (ii), which is subsequently processed into two peptides by protein cleavage. In some embodiments, the nucleic acid sequence encoding (i) is upstream of the nucleic acid sequence encoding (ii), or the nucleic acid sequence encoding (ii) is upstream of the nucleic acid sequence encoding (i). In embodiments, a first promoter controls the expression of the nucleic acid sequence encoding (i), and a second promoter controls the expression of the nucleic acid sequence encoding (ii). In embodiments, the nucleic acid is a plasmid. In embodiments, the nucleic acid includes a viral packaging element. In some embodiments, the disclosure provides cells, such as immunoeffector cells, comprising the nucleic acids described herein, e.g., nucleic acids comprising (i) and (ii) above. Cells may also contain proteases (e.g., endogenous or exogenous) that cleave T2A, P2A, E2A, or F2A cleavage sites.

[0046] In some embodiments, the Disclosure provides a composition comprising, for example, (i) a first nucleic acid encoding a CD19-binding CAR molecule, e.g., a CD19-binding CAR molecule as described herein, e.g., CD19 CAR, and (ii) a second nucleic acid encoding one or more B cell inhibitors, e.g., one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1. In some embodiments, the Disclosure provides a composition comprising, for example, (i) a first nucleic acid encoding a CD19-binding CAR molecule, e.g., a CD19-binding CAR molecule as described herein, e.g., CD19 CAR, and (ii) a CAR molecule that binds to one or more B cell antigens, e.g., CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1. In embodiments, the first nucleic acid and the second nucleic acid each comprise RNA or DNA.

[0047] In some embodiments, the Disclosure provides vectors comprising, for example, one or more nucleic acids as described herein. In certain embodiments, the Disclosure also provides cells comprising vectors or nucleic acids as described herein.

[0048] The Disclosure also provides, in certain embodiments, a composition comprising one or more immunoeffector cells, and (i) a first nucleic acid encoding a CD19-binding CAR molecule, e.g., a CD19-binding CAR molecule as described herein, e.g., CD19 CAR, or a first polypeptide comprising such a CAR molecule, and (ii) a second nucleic acid encoding a CAR molecule binding to one or more B cell antigens, e.g., CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, or a second polypeptide comprising such a CAR molecule. In embodiments, the first nucleic acid or first polypeptide and the second nucleic acid or second polypeptide are each contained within the first immunoeffector cell, e.g., expressed by the first immunoeffector cell. In embodiments, the composition comprises a first immunoeffector cell containing, e.g., the first nucleic acid or first polypeptide, and a second immunoeffector cell containing, e.g., the second nucleic acid or second polypeptide, e.g., expressing the second nucleic acid or second polypeptide. In embodiments, the composition contains both a first nucleic acid or first polypeptide and a second nucleic acid or second polypeptide, for example, an expression, and does not contain cells.

[0049] manufacturing In certain embodiments, the Disclosure provides a method for producing cells, comprising transduction of immunoeffector cells, such as T cells or NK cells, with a vector, such as a CAR-encoding vector, as described herein. In certain embodiments, the Disclosure provides a method for producing cells, comprising introducing a nucleic acid, such as a CAR-encoding nucleic acid, as described herein, into immunoeffector cells, such as T cells or NK cells. In certain embodiments, the Disclosure provides a method for generating a population of RNA-processed cells, comprising introducing in vitro transcribed or synthetic RNA into cells, wherein the RNA includes a nucleic acid, such as a CAR-encoding nucleic acid, as described herein.

[0050] In some embodiments, the production methods disclosed herein further include contacting a population of cells (e.g., CD19 CAR-expressing cells, CD20 CAR-expressing cells, CD22 CAR-expressing cells, B-cell inhibitor cells, or both CD19 CAR-expressing cells and B-cell inhibitor cells) with a nucleic acid encoding a telomerase subunit, such as hTERT. The nucleic acid encoding the telomerase subunit may be DNA.

[0051] In some embodiments, the production method disclosed herein further comprises culturing a population of cells (e.g., CD19 CAR-expressing cells, CD20 CAR-expressing cells, CD22 CAR-expressing cells, B-cell inhibitor cells, or both CD19 CAR-expressing cells and B-cell inhibitor cells) in serum containing 2% hAB serum.

[0052] Symptoms In one embodiment, the disease associated with CD19 expression is selected from proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as spinal cord malformations, myelodysplastic syndromes or preleukemic states, or non-cancer-related signs associated with CD19 expression. In one embodiment, the disease is a solid or humoral tumor. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the disease is a hematological cancer. In one embodiment, the hematological cancer is leukemia. In one embodiment, the cancer is selected from the group consisting of one or more acute leukemias, including but not limited to B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), and acute lymphoblastic leukemia (ALL) (e.g., relapsed and refractory ALL); and one or more chronic leukemias, including but not limited to chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Additional hematological malignancies or conditions include, but are not limited to, mantle cell lymphoma (MCL), B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström macroglobulinemia, and “preleukemic conditions.” Preleukemic conditions encompass a diverse group of hematological conditions that involve the ineffective production (or dysplasia) of blood cells in the bone marrow. In embodiments, diseases associated with CD19 expression include, but are not limited to, atypical and / or non-typical cancers, malignancies, precancerous conditions or proliferative disorders expressing CD19, as well as any combination thereof.

[0053] In one embodiment, the disease associated with CD19 expression is lymphoma, e.g., MCL or Hodgkin lymphoma. In one embodiment, the disease associated with CD19 expression is leukemia, e.g., SLL, CLL and / or ALL.

[0054] In one embodiment, the tumor antigen, for example, the disease associated with the tumor antigen described herein, is selected from proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as spinal cord malformations, myelodysplastic syndromes or preleukemic states, or non-cancerous signs associated with the expression of the tumor antigen described herein. In one embodiment, the disease associated with the tumor antigen described herein is a solid tumor, for example, a solid tumor described herein, such as prostate, colorectal, pancreatic, cervical, stomach, ovarian, head, or lung cancer.

[0055] In one embodiment, the cancer is selected from AML, ALL, B-ALL, T-ALL, B-cell prelymphocytic leukemia, chronic lymphocytic leukemia, CML, hairy cell leukemia, Hodgkin lymphoma, mast cell dysplasia, myelodysplastic syndrome, myeloproliferative neoplasm, plasmacytomyeloma, plasmacytoid dendritic cell neoplasm, or a combination thereof.

[0056] In one embodiment, a subject (e.g., a subject treated with CD19 CAR in appropriate combination with a second agent such as a PD1 inhibitor or PD-L1 inhibitor) is identified as having or possessing at least 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% CD3+ / PD1+ cancer cells, such as DLBCL cells.

[0057] In one embodiment, a subject is identified as having relapsed or having relapsed after treatment with one or more cells expressing a CD19-binding CAR molecule, e.g., CD19 CAR. In another embodiment, a subject is identified as having relapsed or having relapsed based on the reappearance of one or more blast cells in the blood, bone marrow (>5%) or any extramedullary location after a complete response. In yet another embodiment, a subject is identified as having relapsed or having relapsed based on the detection of CD19-blast cells above a predetermined threshold, e.g., 1%, 2%, 3%, 4%, 5%, or 10%.

[0058] CAR therapy In certain embodiments, the treatment method includes CAR therapy, for example, the administration of one or more cells expressing one or more CAR molecules. The cells expressing one or more CAR molecules may be immune effector cells, for example, T cells or NK cells. In certain embodiments, the subject is human.

[0059] In one embodiment, a cell expressing a CAR molecule includes a vector containing a nucleic acid sequence encoding the CAR molecule. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, or retroviral vector. In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further includes a promoter. In one embodiment, the promoter is an EF-1 promoter. In one embodiment, the EF-1 promoter includes the sequence of SEQ ID NO: 100. In one embodiment, the vector is an in vitro transcribed vector, e.g., a vector transcribing the RNA of a nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence in the in vitro vector further includes a poly(A) tail, e.g., a polyA tail described herein, e.g., a polyA tail containing about 150 adenosine bases. In one embodiment, the nucleic acid sequence in the in vitro vector further includes a 3'UTR, e.g., a 3'UTR containing at least one repeat of a 3'UTR derived from human betaglobulin, e.g., a 3'UTR described herein. In one embodiment, the nucleic acid sequence in the in vitro vector further includes a promoter. In one embodiment, the nucleic acid sequence includes a T2A sequence.

[0060] In one embodiment, the cells expressing the CAR molecule are the cells described herein, for example, human T cells or human NK cells, for example, human T cells or human NK cells as described herein. In one embodiment, the human T cells are CD8+ T cells. In one embodiment, the human T cells are CD4+ T cells. In one embodiment, the human T cells are CD4+ / CD8+ T cells. In one embodiment, the human T cells are a mixture of CD8+ T cells and CD4+ T cells. In one embodiment, the cells are autologous T cells. In one embodiment, the cells are allogeneic T cells. In one embodiment, the cells are T cells, and the T cells are diacylglycerol kinase (DGK) deficient. In one embodiment, the cells are T cells, and the T cells are Ikaros deficient. In one embodiment, the cells are T cells, and the T cells are both DGK and Ikaros deficient.

[0061] In another embodiment, cells expressing a CAR molecule, such as the CAR molecule described herein, may further express another drug, such as a drug that enhances the activity of the CAR-expressing cell.

[0062] In one embodiment, the method comprises administering cells expressing CAR molecules as described herein in combination with an agent that enhances the activity of the CAR-expressing cells, wherein the agent is a cytokine, such as IL-7, IL-15, IL-21, or a combination thereof. The cytokine can be delivered in combination with the administration of the CAR-expressing cells, for example, simultaneously with or immediately after the administration. Alternatively, the cytokine can be delivered long after the administration of the CAR-expressing cells, for example, after assessment of the target response to the CAR-expressing cells.

[0063] For example, in one embodiment, the agent that enhances the activity of CAR-expressing cells may be an agent that inhibits immunosuppressive molecules. Examples of immunosuppressive molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the agent that inhibits immunosuppressive molecules includes a second polypeptide that provides a positive signal to the cell, a first polypeptide associated with an intracellular signaling domain, e.g., an inhibitor molecule, e.g. In one embodiment, the drug comprises a first polypeptide of an immunoinhibitory molecule, e.g., PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein [e.g., a co-stimulatory domain (e.g., 41BB, CD27, or CD28) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein)]. In one embodiment, the drug comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein).

[0064] In one embodiment, a lymphocyte infusion, for example, an allogeneic lymphocyte infusion containing at least one type of CD19 CAR-expressing cell as described herein, and which may contain at least one cell expressing a CAR directed toward a B cell antigen, is used for the treatment of cancer. In one embodiment, an autologous lymphocyte infusion, containing at least one type of CD19-expressing cell, and which may contain at least one cell expressing a CAR directed toward a B cell antigen, is used for the treatment of cancer.

[0065] In one embodiment, CAR-expressing cells, such as T cells, are administered to subjects who have undergone prior stem cell transplantation, such as autologous stem cell transplantation, or to subjects who have received prior melphalan doses.

[0066] In one embodiment, cells expressing a CAR molecule, for example, a CAR molecule as described herein, are administered in combination with a drug that improves one or more side effects associated with the administration of cells expressing the CAR molecule or with the administration of a B cell inhibitor, for example, a drug as described herein.

[0067] In one embodiment, a CAR molecule, such as a cell expressing the CD19 CAR molecule described herein, and a B cell inhibitor are administered in combination with additional agents that treat a CD19-related disease, such as additional agents described herein.

[0068] In one embodiment, cells expressing a CAR molecule, for example, a CAR molecule described herein, are administered in the dose and / or administration schedule described herein.

[0069] In one embodiment, the CAR molecule is introduced into T cells, for example, using in vitro transcription, and the subject (e.g., human) receives an initial dose of cells containing the CAR molecule, and one or more subsequent doses of cells containing the CAR molecule, the one or more subsequent doses being administered less than 15 days after the initial dose, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the initial dose. In one embodiment, more than one dose of cells containing the CAR molecule is administered to the subject (e.g., human) weekly, for example, two, three, or four doses of cells containing the CAR molecule are administered weekly. In one embodiment, the subject (e.g., human subject) receives more than one weekly dose of cells containing the CAR molecule (e.g., two, three, or four weekly doses) (also referred to herein as a cycle), followed by a week without administration of cells containing the CAR molecule, and then one or more further doses of cells containing the CAR molecule (e.g., more than one weekly dose of cells containing the CAR molecule) are administered to the subject. In another embodiment, a subject (e.g., a human subject) receives cells containing CAR molecules for one or more cycles, with the time between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, cells containing CAR molecules are administered every other day, three times a week. In one embodiment, cells containing CAR molecules are administered for at least 2, 3, 4, 5, 6, 7, or 8 weeks, or longer.

[0070] In one embodiment, the treatments described herein (e.g., CD20 CAR therapy, CD22 CAR therapy, or a combination of a B cell inhibitor and a CD19 CAR molecule, e.g., cells expressing the CD19 CAR molecule as described herein) are administered as first-line treatment for a disease, e.g., cancer, e.g., cancer as described herein. In another embodiment, the treatments described herein (e.g., CD20 CAR therapy, CD22 CAR therapy, or a combination of a B cell inhibitor and a CD19 CAR molecule, e.g., cells expressing the CD19 CAR molecule as described herein) are administered as second, third, or fourth-line treatments for a disease, e.g., cancer, e.g., cancer as described herein.

[0071] In one embodiment, a population of cells described herein is administered. In some embodiments, the population of cells is isolated or purified.

[0072] In one embodiment, the method comprises administering a population of cells, the majority of which contain the CAR molecules described herein. In some embodiments, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of CAR-expressing cells may comprise a first cell expressing a CAR having an anti-CD19 binding domain as described herein, and a second cell expressing a CAR having a different B-cell antigen-binding domain. In embodiments, the first and second cell populations are T cells. In embodiments, the first and second populations of T cells are of the same isotype, for example, both being CD4+ T cells or both being CD8+ T cells. In other embodiments, the first and second populations of T cells are of different isotypes, for example, the first population includes CD4+ T cells and the second population includes CD8+ T cells. In embodiments, the first and second populations of T cells are cell types described in WO2012 / 129514, which is incorporated herein by reference in its entirety. As an alternative example, a population of cells may include a single cell type that expresses both CARs having an anti-CD19 binding domain as described herein and CARs having different B-cell antigen-binding domains. As an alternative example, a population of cells may include a single cell type that expresses CARs having two or more (e.g., two, three, four, or five) B-cell antigen-binding domains, such as bispecific CARs, such as those described herein. As an alternative example, a population of CAR-expressing cells may include a first cell expressing a CAR containing an anti-CD19 binding domain, such as the anti-CD19 binding domain described herein, and a second cell expressing a CAR containing an antigen-binding domain to a target other than CD19 (e.g., CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, CD79a, or mesoserine). In one embodiment, a population of CAR-expressing cells may include, for example, a first CAR-expressing cell containing a primary intracellular signaling domain and a second CAR-expressing cell containing a secondary signaling domain.In one embodiment, the population of CAR-expressing cells includes, for example, a first cell expressing a CAR containing a first secondary signaling domain, and a second cell expressing a CAR containing a secondary signaling domain different from the first secondary signaling domain.

[0073] For example, if the first B cell inhibitor is CD19 CAR-expressing cells and the second B cell inhibitor is CD10 CAR-expressing cells, the first and second CARs may be expressed by the same cell type or by different cell types. For example, in some embodiments, the cells expressing the CD19 CAR are CD4+ T cells and the cells expressing the CD10 CAR are CD8+ T cells, or the cells expressing the CD19 CAR are CD8+ T cells and the cells expressing the CD10 CAR are CD4+ T cells. In other embodiments, the cells expressing the CD19 CAR are T cells and the cells expressing the CD10 CAR are NK cells, or the cells expressing the CD19 CAR are NK cells and the cells expressing the CD10 CAR are T cells. In other embodiments, both the cells expressing the CD19 CAR and the cells expressing the CD10 CAR are NK cells, or both are T cells, for example, both are CD4+ T cells, or both are CD8+ T cells. In yet another embodiment, a single cell expresses both a CD19 CAR and a CD10 CAR, which is, for example, an NK cell or a T cell, such as a CD4+ T cell or a CD8+ T cell. The first and second CARs may contain the same intracellular signaling domain or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR contains a CD3 zeta signaling domain and the CD10 CAR contains a co-stimulatory domain, such as 41BB, CD27, or CD28; on the other hand, in some embodiments, the CD19 CAR contains a co-stimulatory domain, such as 41BB, CD27, or CD28, and the CD10 CAR contains a CD3 zeta signaling domain.In other embodiments, each of the CD19 CAR and CD10 CAR contains the same type of primary signaling domain, e.g., a CD3 zeta signaling domain, but the CD19 CAR and CD10 CAR contain different co-stimulatory domains, for example, (1) the CD19 CAR contains a 41BB co-stimulatory domain and the CD10 CAR contains a different co-stimulatory domain, e.g., a CD27 co-stimulatory domain; (2) the CD19 CAR contains a CD27 co-stimulatory domain and the CD10 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (3) the CD19 CAR contains a 41BB co-stimulatory domain and the CD10 CAR contains a CD28 co-stimulatory domain; (4) the CD19 CAR contains a CD28 co-stimulatory domain and the CD10 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (5) the CD19 CAR contains a CD27 co-stimulatory domain and the CD10 The CAR contains a CD28 costimulatory domain, or (6) a CD19 CAR contains a CD28 costimulatory domain, and a CD10 CAR contains a CD27 costimulatory domain. In another embodiment, the cell contains a CAR that contains both a CD19 antigen-binding domain and a CD10 antigen-binding domain, for example, a bispecific antibody.

[0074] As another example, if the first B cell inhibitor is CD19 CAR-expressing cells and the second B cell inhibitor is CD20 CAR-expressing cells, the first and second CARs may be expressed by the same cell type or by different cell types. For example, in some embodiments, the cells expressing the CD19 CAR are CD4+ T cells and the cells expressing the CD20 CAR are CD8+ T cells, or the cells expressing the CD19 CAR are CD8+ T cells and the cells expressing the CD20 CAR are CD4+ T cells. In other embodiments, the cells expressing the CD19 CAR are T cells and the cells expressing the CD20 CAR are NK cells, or the cells expressing the CD19 CAR are NK cells and the cells expressing the CD20 CAR are T cells. In other embodiments, cells expressing the CD19 CAR and cells expressing the CD20 CAR are both NK cells or both are T cells, for example, both are CD4+ T cells or both are CD8+ T cells. In yet another embodiment, a single cell expresses both the CD19 CAR and the CD20 CAR, and this cell is, for example, an NK cell or a T cell, for example, a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR may contain the same intracellular signaling domain or may contain different intracellular signaling domains. For example, in some embodiments, the CD19 CAR contains a CD3 zeta signaling domain and the CD20 CAR contains a co-stimulatory domain, for example, 41BB, CD27, or CD28 co-stimulatory domain, while in other embodiments, the CD19 CAR contains a co-stimulatory domain, for example, 41BB, CD27, or CD28 co-stimulatory domain and the CD20 CAR contains a CD3 zeta signaling domain.In other embodiments, each of the CD19 CAR and CD20 CAR contains the same type of primary signaling domain, e.g., a CD3 zeta signaling domain, but the CD19 CAR and CD20 CAR contain different co-stimulatory domains, for example, (1) the CD19 CAR contains a 41BB co-stimulatory domain and the CD20 CAR contains a different co-stimulatory domain, e.g., a CD27 co-stimulatory domain; (2) the CD19 CAR contains a CD27 co-stimulatory domain and the CD20 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (3) the CD19 CAR contains a 41BB co-stimulatory domain and the CD20 CAR contains a CD28 co-stimulatory domain; (4) the CD19 CAR contains a CD28 co-stimulatory domain and the CD20 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (5) the CD19 CAR contains a CD27 co-stimulatory domain and the CD20 The CAR contains a CD28 costimulatory domain, or (6) a CD19 CAR contains a CD28 costimulatory domain and a CD20 CAR contains a CD27 costimulatory domain. In another embodiment, the cell contains a CAR that contains both a CD19 antigen-binding domain and a CD20 antigen-binding domain, for example, a bispecific antibody.

[0075] As another example, if the first B cell inhibitor is CD19 CAR-expressing cells and the second B cell inhibitor is CD22 CAR-expressing cells, the first and second CARs may be expressed by the same cell type or by different cell types. For example, in some embodiments, the cells expressing the CD19 CAR are CD4+ T cells and the cells expressing the CD22 CAR are CD8+ T cells, or the cells expressing the CD19 CAR are CD8+ T cells and the cells expressing the CD22 CAR are CD4+ T cells. In other embodiments, the cells expressing the CD19 CAR are T cells and the cells expressing the CD22 CAR are NK cells, or the cells expressing the CD19 CAR are NK cells and the cells expressing the CD22 CAR are T cells. In other embodiments, both the cells expressing the CD19 CAR and the cells expressing the CD22 CAR are NK cells, or both are T cells, for example, both are CD4+ T cells, or both are CD8+ T cells. In yet another embodiment, a single cell expresses both a CD19 CAR and a CD22 CAR, which is, for example, an NK cell or a T cell, such as a CD4+ T cell or a CD8+ T cell. The first and second CARs may contain the same intracellular signaling domain or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR contains a CD3 zeta signaling domain and the CD22 CAR contains a co-stimulatory domain, such as 41BB, CD27, or CD28; on the other hand, in some embodiments, the CD19 CAR contains a co-stimulatory domain, such as 41BB, CD27, or CD28, and the CD22 CAR contains a CD3 zeta signaling domain.In other embodiments, each of the CD19 CAR and CD22 CAR includes the same type of primary signaling domain, e.g., a CD3 zeta signaling domain, but the CD19 CAR and CD22 CAR include different co-stimulatory domains, for example: (1) the CD19 CAR includes a 41BB co-stimulatory domain and the CD22 CAR includes a different co-stimulatory domain, e.g., a CD27 co-stimulatory domain; (2) the CD19 CAR includes a CD27 co-stimulatory domain and the CD22 CAR includes a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (3) the CD19 CAR includes a 41BB co-stimulatory domain and the CD22 CAR includes a CD28 co-stimulatory domain; (4) the CD19 CAR includes a CD28 co-stimulatory domain and the CD22 CAR includes a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (5) the CD19 CAR includes a CD27 co-stimulatory domain and the CD22 The CAR contains a CD28 costimulatory domain, or (6) a CD19 CAR contains a CD28 costimulatory domain and a CD22 CAR contains a CD27 costimulatory domain. In another embodiment, the cell contains a CAR that contains both a CD19 antigen-binding domain and a CD22 antigen-binding domain, for example, a bispecific antibody.

[0076] As another example, if the first B cell inhibitor is CD19 CAR-expressing cells and the second B cell inhibitor is CD34 CAR-expressing cells, the first and second CARs may be expressed by the same cell type or by different cell types. For example, in some embodiments, the cells expressing the CD19 CAR are CD4+ T cells and the cells expressing the CD34 CAR are CD8+ T cells, or the cells expressing the CD19 CAR are CD8+ T cells and the cells expressing the CD34 CAR are CD4+ T cells. In other embodiments, the cells expressing the CD19 CAR are T cells and the cells expressing the CD34 CAR are NK cells, or the cells expressing the CD19 CAR are NK cells and the cells expressing the CD34 CAR are T cells. In other embodiments, both the cells expressing the CD19 CAR and the cells expressing the CD34 CAR are NK cells, or both are T cells, for example, both are CD4+ T cells, or both are CD8+ T cells. In yet another embodiment, a single cell expresses both a CD19 CAR and a CD34 CAR, which is, for example, an NK cell or a T cell, such as a CD4+ T cell or a CD8+ T cell. The first and second CARs may contain the same intracellular signaling domain or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR contains a CD3 zeta signaling domain and the CD34 CAR contains a co-stimulatory domain, such as 41BB, CD27, or CD28; on the other hand, in some embodiments, the CD19 CAR contains a co-stimulatory domain, such as 41BB, CD27, or CD28, and the CD34 CAR contains a CD3 zeta signaling domain.In other embodiments, each of the CD19 CAR and CD34 CAR contains the same type of primary signaling domain, e.g., a CD3 zeta signaling domain, but the CD19 CAR and CD34 CAR contain different co-stimulatory domains, for example: (1) the CD19 CAR contains a 41BB co-stimulatory domain and the CD34 CAR contains a different co-stimulatory domain, e.g., a CD27 co-stimulatory domain; (2) the CD19 CAR contains a CD27 co-stimulatory domain and the CD34 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (3) the CD19 CAR contains a 41BB co-stimulatory domain and the CD34 CAR contains a CD28 co-stimulatory domain; (4) the CD19 CAR contains a CD28 co-stimulatory domain and the CD34 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (5) the CD19 CAR contains a CD27 co-stimulatory domain and the CD34 The CAR contains a CD28 costimulatory domain, or (6) a CD19 CAR contains a CD28 costimulatory domain and a CD34 CAR contains a CD27 costimulatory domain. In another embodiment, the cell contains a CAR that contains both a CD19 antigen-binding domain and a CD34 antigen-binding domain, for example, a bispecific antibody.

[0077] As another example, if the first B cell inhibitor is CD19 CAR-expressing cells and the second B cell inhibitor is CD123 CAR-expressing cells, the first and second CARs may be expressed by the same cell type or by different cell types. For example, in some embodiments, the cells expressing the CD19 CAR are CD4+ T cells and the cells expressing the CD123 CAR are CD8+ T cells, or the cells expressing the CD19 CAR are CD8+ T cells and the cells expressing the CD123 CAR are CD4+ T cells. In other embodiments, the cells expressing the CD19 CAR are T cells and the cells expressing the CD123 CAR are NK cells, or the cells expressing the CD19 CAR are NK cells and the cells expressing the CD123 CAR are T cells. In other embodiments, cells expressing the CD19 CAR and cells expressing the CD123 CAR are both NK cells or both are T cells, for example, both are CD4+ T cells or both are CD8+ T cells. In yet another embodiment, a single cell expresses both the CD19 CAR and the CD123 CAR, and this cell is, for example, an NK cell or a T cell, for example, a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR may contain the same intracellular signaling domain or may contain different intracellular signaling domains. For example, in some embodiments, the CD19 CAR contains a CD3 zeta signaling domain and the CD123 CAR contains a co-stimulatory domain, for example, 41BB, CD27, or CD28, while in other embodiments, the CD19 CAR contains a co-stimulatory domain, for example, 41BB, CD27, or CD28, and the CD123 CAR contains a CD3 zeta signaling domain.In other embodiments, each of the CD19 CAR and CD123 CAR contains the same type of primary signaling domain, e.g., a CD3 zeta signaling domain, but the CD19 CAR and CD123 CAR contain different co-stimulatory domains, for example: (1) the CD19 CAR contains a 41BB co-stimulatory domain and the CD123 CAR contains a different co-stimulatory domain, e.g., a CD27 co-stimulatory domain; (2) the CD19 CAR contains a CD27 co-stimulatory domain and the CD123 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (3) the CD19 CAR contains a 41BB co-stimulatory domain and the CD123 CAR contains a CD28 co-stimulatory domain; (4) the CD19 CAR contains a CD28 co-stimulatory domain and the CD123 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (5) the CD19 CAR contains a CD27 co-stimulatory domain and the CD123 The CAR contains a CD28 costimulatory domain, or (6) a CD19 CAR contains a CD28 costimulatory domain, and a CD123 CAR contains a CD27 costimulatory domain. In another embodiment, the cell contains a CAR that contains both a CD19 antigen-binding domain and a CD123 antigen-binding domain, for example, a bispecific antibody.

[0078] As another example, if the first B cell inhibitor is CD19 CAR-expressing cells and the second B cell inhibitor is FLT-3 CAR-expressing cells, the first and second CARs may be expressed by the same cell type or by different cell types. For example, in some embodiments, the cells expressing the CD19 CAR are CD4+ T cells and the cells expressing the FLT-3 CAR are CD8+ T cells, or the cells expressing the CD19 CAR are CD8+ T cells and the cells expressing the FLT-3 CAR are CD4+ T cells. In other embodiments, the cells expressing the CD19 CAR are T cells and the cells expressing the FLT-3 CAR are NK cells, or the cells expressing the CD19 CAR are NK cells and the cells expressing the FLT-3 CAR are T cells. In other embodiments, cells expressing the CD19 CAR and cells expressing the FLT-3 CAR are both NK cells or both are T cells, for example, both are CD4+ T cells or both are CD8+ T cells. In yet another embodiment, a single cell expresses both the CD19 CAR and the FLT-3 CAR, and this cell is, for example, an NK cell or a T cell, for example, a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR may contain the same intracellular signaling domain or may contain different intracellular signaling domains. For example, in some embodiments, the CD19 CAR contains a CD3 zeta signaling domain and the FLT-3 CAR contains a co-stimulatory domain, for example, 41BB, CD27, or CD28 co-stimulatory domain, while in other embodiments, the CD19 CAR contains a co-stimulatory domain, for example, 41BB, CD27, or CD28 co-stimulatory domain and the FLT-3 CAR contains a CD3 zeta signaling domain.In other embodiments, each of the CD19 CAR and FLT-3 CAR contains the same type of primary signaling domain, e.g., a CD3 zeta signaling domain, but the CD19 CAR and FLT-3 CAR contain different co-stimulatory domains, for example, (1) the CD19 CAR contains a 41BB co-stimulatory domain and the FLT-3 CAR contains a different co-stimulatory domain, e.g., a CD27 co-stimulatory domain; (2) the CD19 CAR contains a CD27 co-stimulatory domain and the FLT-3 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (3) the CD19 CAR contains a 41BB co-stimulatory domain and the FLT-3 CAR contains a CD28 co-stimulatory domain; (4) the CD19 CAR contains a CD28 co-stimulatory domain and the FLT-3 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (5) the CD19 CAR contains a CD27 co-stimulatory domain and the FLT-3 The CAR contains a CD28 costimulatory domain, or (6) a CD19 CAR contains a CD28 costimulatory domain and an FLT-3 CAR contains a CD27 costimulatory domain. In another embodiment, the cell contains a CAR that contains both a CD19 antigen-binding domain and an FLT-3 antigen-binding domain, for example, a bispecific antibody.

[0079] As another example, if the first B cell inhibitor is CD19 CAR-expressing cells and the second B cell inhibitor is ROR1 CAR-expressing cells, the first and second CARs may be expressed by the same cell type or by different cell types. For example, in some embodiments, the cells expressing the CD19 CAR are CD4+ T cells and the cells expressing the ROR1 CAR are CD8+ T cells, or the cells expressing the CD19 CAR are CD8+ T cells and the cells expressing the ROR1 CAR are CD4+ T cells. In other embodiments, the cells expressing the CD19 CAR are T cells and the cells expressing the ROR1 CAR are NK cells, or the cells expressing the CD19 CAR are NK cells and the cells expressing the ROR1 CAR are T cells. In other embodiments, both the cells expressing the CD19 CAR and the cells expressing the ROR1 CAR are NK cells, or both are T cells, for example, both are CD4+ T cells, or both are CD8+ T cells. In yet another embodiment, a single cell expresses both the CD19 CAR and the ROR1 CAR, which is, for example, an NK cell or a T cell, such as a CD4+ T cell or a CD8+ T cell. The first and second CARs may contain the same intracellular signaling domain or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR contains a CD3 zeta signaling domain and the ROR1 CAR contains a co-stimulatory domain, such as 41BB, CD27, or CD28; on the other hand, in some embodiments, the CD19 CAR contains a co-stimulatory domain, such as 41BB, CD27, or CD28, and the ROR1 CAR contains a CD3 zeta signaling domain.In other embodiments, each of the CD19 CAR and ROR1 CAR contains the same type of primary signaling domain, e.g., a CD3 zeta signaling domain, but the CD19 CAR and ROR1 CAR contain different co-stimulatory domains, for example, (1) the CD19 CAR contains a 41BB co-stimulatory domain and the ROR1 CAR contains a different co-stimulatory domain, e.g., a CD27 co-stimulatory domain; (2) the CD19 CAR contains a CD27 co-stimulatory domain and the ROR1 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (3) the CD19 CAR contains a 41BB co-stimulatory domain and the ROR1 CAR contains a CD28 co-stimulatory domain; (4) the CD19 CAR contains a CD28 co-stimulatory domain and the ROR1 CAR contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (5) the CD19 CAR contains a CD27 co-stimulatory domain and the ROR1 The CAR contains a CD28 costimulatory domain, or (6) a CD19 CAR contains a CD28 costimulatory domain and a ROR1 CAR contains a CD27 costimulatory domain. In another embodiment, the cell contains a CAR that contains both a CD19 antigen-binding domain and a ROR1 antigen-binding domain, for example, a bispecific antibody.

[0080] More generally, if the first B cell inhibitor contains a CD19 CAR and there is a second B cell inhibitor, for example, a second B cell inhibitor containing a second CAR, the first CAR and the second B cell inhibitor may be expressed by the same cell type or by different cell types. For example, in some embodiments, the cells expressing the CD19 CAR are CD4+ T cells and the cells expressing the second B cell inhibitor are CD8+ T cells, or the cells expressing the CD19 CAR are CD8+ T cells and the cells expressing the second B cell inhibitor are CD4+ T cells. In other embodiments, the cells expressing the CD19 CAR are T cells and the cells expressing the second B cell inhibitor are NK cells, or the cells expressing the CD19 CAR are NK cells and the cells expressing the second B cell inhibitor are T cells. In other embodiments, the cells expressing the CD19 CAR and the cells expressing the second B cell inhibitor are both NK cells or both are T cells, for example, both are CD4+ T cells or both are CD8+ T cells. In yet another embodiment, a single cell expresses both the CD19 CAR and the second B cell inhibitor, and this cell is, for example, an NK cell or a T cell, for example, a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR may contain the same intracellular signaling domain or may contain different intracellular signaling domains. For example, in some embodiments, the CD19 CAR contains a CD3 zeta signaling domain and the second B cell inhibitor (or CAR) contains a co-stimulatory domain, for example, 41BB, CD27, or CD28 co-stimulatory domain, while in other embodiments, the CD19 CAR contains a co-stimulatory domain, for example, 41BB, CD27, or CD28 co-stimulatory domain and the second B cell inhibitor (or second CAR) contains a CD3 zeta signaling domain.In other embodiments, each of the CD19 CAR and the second B cell inhibitor (or second CAR) contains the same type of primary signaling domain, e.g., a CD3 zeta signaling domain, but the CD19 CAR and the second B cell inhibitor contain different co-stimulatory domains, for example, (1) the CD19 CAR contains a 41BB co-stimulatory domain and the second B cell inhibitor (or second CAR) contains a different co-stimulatory domain, e.g., a CD27 co-stimulatory domain; (2) the CD19 CAR contains a CD27 co-stimulatory domain and the second B cell inhibitor (or second CAR) contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (3) the CD19 CAR contains a 41BB co-stimulatory domain and the second B cell inhibitor (or second CAR) contains a CD28 co-stimulatory domain; (4) the CD19 CAR contains a CD28 co-stimulatory domain and the second B cell inhibitor (or second CAR) contains a different co-stimulatory domain, e.g., a 41BB co-stimulatory domain; (5) CD19 (6) The CAR contains a CD27 costimulatory domain, and the second B cell inhibitor (or second CAR) contains a CD28 costimulatory domain, or the CD19 CAR contains a CD28 costimulatory domain, and the second B cell inhibitor (or second CAR) contains a CD27 costimulatory domain. In another embodiment, the cell contains a CAR that contains both a CD19 antigen-binding domain and an antigen-binding domain directed to a second antigen, for example, a bispecific antibody.

[0081] In one embodiment, the 4-1BB co-stimulatory domain includes the sequence of SEQ ID NO: 16. In one embodiment, the 4-1BB co-stimulatory domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but 20, 10, or five or fewer modifications (e.g., substitutions), of the amino acid sequence of SEQ ID NO: 16, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the 4-1BB co-stimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO: 60, or a sequence having 95-99% identity with it.

[0082] In one embodiment, the CD27 costimulatory domain includes the sequence of SEQ ID NO: 16. In one embodiment, the CD27 costimulatory domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 16, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 16, but with 20, 10, or 5 or fewer modifications (e.g., substitutions). In one embodiment, the CD27 costimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO: 17, or a sequence having 95-99% identity with it.

[0083] In one embodiment, the CD28 co-stimulatory domain includes the sequence of SEQ ID NO: 1317. In one embodiment, the CD28 co-stimulatory domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but 20, 10, or five or fewer modifications (e.g., substitutions), of the amino acid sequence of SEQ ID NO: 1317, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 1317. In one embodiment, the CD28 co-stimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO: 1318, or a sequence having 95-99% identity with it.

[0084] In one embodiment, the wild-type ICOS co-stimulatory domain includes the sequence of SEQ ID NO: 1319. In one embodiment, the wild-type ICOS co-stimulatory domain includes the amino acid sequence of SEQ ID NO: 1319, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 1319, with at least 1, 2, or 3 modifications (e.g., substitutions), but 20, 10, or 5 or fewer modifications (e.g., substitutions). In one embodiment, the wild-type ICOS co-stimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO: 1320, or a sequence having 95-99% identity with it.

[0085] In one embodiment, the Y→F mutant ICOS costimulatory domain includes the sequence of SEQ ID NO: 1321. In one embodiment, the Y→F mutant ICOS costimulatory domain includes the amino acid sequence of SEQ ID NO: 1321, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 1321, with at least 1, 2, or 3 modifications (e.g., substitutions), but 20, 10, or 5 or fewer modifications (e.g., substitutions). In one embodiment, the Y→F mutant ICOS costimulatory domain is encoded by a nucleic acid sequence having 95-99% identity with the nucleic acid sequence of SEQ ID NO: 1320 (where SEQ ID NO: 1320 codes for wild-type ICOS).

[0086] In the embodiment, the primary signaling domain includes a functional signaling domain of CD3 zeta. In the embodiment, the functional signaling domain of CD3 zeta includes SEQ ID NO: 17 (mutant CD3 zeta) or SEQ ID NO: 43 (wild-type human CD3 zeta).

[0087] In one embodiment, the method comprises administering a population of cells in which at least one type of cell expresses a CAR, for example, a CAR having an anti-CD19 domain as described herein, and a second cell expressing a drug that enhances the activity of the CAR-expressing cells, for example, a drug that enhances the activity of the CAR-expressing cells. For example, in one embodiment, the drug may be a drug that inhibits an immunosuppressive molecule. Examples of immunosuppressive molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the drug that inhibits an immunosuppressive molecule includes a second polypeptide that provides a positive signal to the cell, for example, a first polypeptide associated with an intracellular signaling domain as described herein, e.g., an inhibitory molecule. In one embodiment, the drug comprises a first polypeptide of an inhibitory molecule, e.g., PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein [e.g., a co-stimulatory domain (e.g., 41BB, CD27, or CD28 as described herein) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein)]. In one embodiment, the drug comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein).

[0088] In one embodiment, the B cell inhibitor comprises one or more inhibitors of CD10, CD19, CD20, CD22, CD34, FLT-3, or ROR1. In one embodiment, the B cell inhibitor comprises an effective number of one or more cells expressing a CAR molecule that binds to one or more of CD10, CD20, CD22, CD34, FLT-3, ROR1, CD79b, CD179b, or CD79a. In one embodiment, the B cell inhibitor comprises a CD123 CAR. In one embodiment, the B cell inhibitor comprises one or more cells expressing a CAR molecule that binds to CD123. In one embodiment, the disease is a CD19-negative cancer, e.g., a CD19-negative recurrent cancer. In one embodiment, CD19 CAR-expressing cells are administered simultaneously with, before, or after, one or more B cell inhibitors.

[0089] In one embodiment, the method further comprises administering a CD19 inhibitor, e.g., CD19 CAR-expressing cells. In one embodiment, the CD19 inhibitor comprises a CD19 CAR, and the B-cell inhibitor comprises a CD123 CAR. In one embodiment, the CD19 CAR or CD123 CAR comprises a divided intracellular signaling domain such that when both the CD19 CAR and the CD123 CAR bind to target cells, e.g., target CD19+CD123+ cells (e.g., B-ALL blast cells), complete activation of cells, e.g., an immune effector cell population, occurs compared to the activation when the CD19 CAR and the CD123 CAR bind to target cells expressing either CD19 or CD123 (e.g., hematopoietic stem cells). In one embodiment, the CD123 CAR comprises a 4-1BB signaling domain, and the CD19 CAR comprises a CD3 zeta signaling domain. In one embodiment, the CD123CAR includes a co-stimulatory domain, e.g., a 4-1BB signaling domain, and the CD19CAR includes a primary signaling domain, e.g., a CD3 zeta signaling domain. In one embodiment, the CD123CAR includes a primary signaling domain, e.g., a CD3 zeta signaling domain, and the CD19CAR includes a co-stimulatory domain, e.g., a 4-1BB signaling domain. In one embodiment, the B cell inhibitor includes a CAR containing a co-stimulatory domain (e.g., a CAR directed to CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a), and the CD19CAR includes a primary signaling domain. In one embodiment, the B cell inhibitor comprises a CAR containing a primary signaling domain (e.g., a CAR directed to CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a), and the CD19 CAR comprises a co-stimulatory domain. In one embodiment, the B cell inhibitor comprises one or more cells expressing a CAR molecule that binds to CD123, and the CD19 CAR-expressing cells are administered concurrently with the B cell inhibitor.In one embodiment, CD123CAR includes a 4-1BB signaling domain, and CD19CAR includes a CD3 zeta signaling domain.

[0090] In one embodiment, the method further includes transplanting cells, such as hematopoietic stem cells, or bone marrow, into a mammal.

[0091] In another embodiment, the present invention relates to cells expressing a CAR molecule as described herein, such as the CD19 CAR molecule, for use as a pharmaceutical in combination with a B cell inhibitor, such as the B cell inhibitor described herein.

[0092] In another embodiment, the present invention relates to cells expressing a CAR molecule as described herein, such as the CD19 CAR molecule, for use in combination with a B cell inhibitor, such as the B cell inhibitor described herein, in the treatment of a CD19-expressing disease. In another embodiment, the present invention relates to a B cell inhibitor as described herein, for use in combination with cells expressing a CAR molecule as described herein, such as the CD19 CAR molecule, in the treatment of a CD19-expressing disease. In another embodiment, the present invention relates to cells expressing a CAR molecule as described herein, such as the CD19 CAR molecule, for use in combination with a B cell inhibitor, such as the B cell inhibitor described herein, in the treatment of cancer, such as the cancer described herein.

[0093] In one embodiment, the method comprises administering a population of cells in which at least one type of cell expresses the therapeutic agent described herein (e.g., a CD20 CAR, a CD22 CAR, or a CAR having an anti-CD19 domain as described herein in combination with a B-cell inhibitor), and an agent that enhances the activity of the CAR-expressing cells, wherein the agent is a cytokine, e.g., IL-7, IL-15, IL-21, or a combination thereof. The cytokine can be delivered in combination with the administration of the CAR-expressing cells, for example, simultaneously with or immediately after such administration. Alternatively, the cytokine can be delivered long after the administration of the CAR-expressing cells, for example, after assessment of the target response to the CAR-expressing cells. Related compositions for use in producing pharmaceuticals and methods for producing pharmaceuticals are also provided.

[0094] In one embodiment, the cells described herein (for example, cells expressing the CD20 CAR molecule, cells expressing the CD22 CAR molecule, or cells expressing the CD19 CAR molecule, for example the CD19 CAR molecule described herein, in combination with a B cell inhibitor) are administered in combination with a drug that enhances the efficacy of the cells expressing the CAR molecule or one of the efficacy of the inhibitor, for example, a drug described herein.

[0095] In one embodiment, the cells described herein (for example, cells expressing the CD20 CAR molecule, cells expressing the CD22 CAR molecule, or cells expressing the CD19 CAR molecule, for example the CD19 CAR molecule described herein, in combination with a B cell inhibitor) are administered in combination with a drug that improves one or more side effects associated with the administration of the cells expressing the CAR molecule or with the administration of one of the inhibitors, for example, a drug described herein.

[0096] In one embodiment, cells expressing the CD19 CAR molecule, for example, the CD19 CAR molecule described herein, are administered in combination with a B-cell inhibitor and a drug for treating Hodgkin lymphoma, for example, the drug described herein.

[0097] In some embodiments, the Disclosure provides a method for treating a patient who is unresponsive, partially responsive, or relapsed to a CD19 inhibitor, e.g., CD19 CAR therapy, comprising administering to the patient one or more (e.g., two, three, four, five, six, seven, eight, nine, or all) inhibitors of B cell inhibitors, e.g., B cell inhibitors described herein, e.g., two, three, four, five, six, seven, eight, nine, or all of them, CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1. In one embodiment, the B cell inhibitor is a CAR-expressing cell (e.g., a T cell or NK cell), which is one or more (e.g., two, three, four, five, six, or all of them) inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1. In embodiments, a patient has or is identified as having cancer cells that are positive for CD19-negative cancer cells and one or more (e.g., two, three, four, five, six, seven, eight, nine, or all) of the following: CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. In embodiments, the method further comprises administering to the patient a B-cell inhibitor that is positive for cancer cells, for example, an inhibitor of one or more (e.g., two, three, four, five, six, seven, eight, nine, or all) of the following that is positive for cancer cells: CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. In embodiments, the method further includes one or both of the steps of determining whether a patient has CD19-negative cancer cells, and determining whether a patient has cancer cells that are positive for one or more of the following (e.g., two, three, four, five, six, seven, eight, nine, or all) CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.In the embodiment, the subject is identified as having, or possessing, a population of tumors or cancer cells that are negative for CD19 expression in a test measured by binding to an anti-CD19 antibody, for example, an antibody having the same specificity as any of the CAR molecules in Table 2 or Table 3.

[0098] In another embodiment, the present invention features a composition comprising cells expressing a chimeric antigen receptor (CAR) molecule that binds to CD19, in combination with a B cell inhibitor, for example, a B cell inhibitor selected from inhibitors of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, or a combination thereof. The CAR-expressing cells and the B cell inhibitor may exist in a single dosage form or in two or more dosage forms.

[0099] In one embodiment, the composition is a pharmaceutically acceptable composition.

[0100] In embodiments, the compositions disclosed herein (e.g., nucleic acids, vectors, or cells) are intended for use as pharmaceuticals.

[0101] In embodiments, the compositions disclosed herein are for use in the treatment of diseases associated with the expression of B cell antigens (e.g., CD19), such as B cell leukemia or lymphoma.

[0102] CD19 inhibitors In embodiments, the CD19 inhibitor is a small molecule, an antibody, an antibody fragment, or a cell therapy drug.

[0103] In some embodiments, a CD19 inhibitor (e.g., a cell therapy drug or antibody) is administered in combination with one or more B cell inhibitors, such as one or more inhibitors of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, or is present in the composition together with such B cell inhibitors.

[0104] In one embodiment, cells express a CAR molecule comprising an anti-CD19 binding domain (e.g., a mouse or humanized antibody or antibody fragment that specifically binds to CD19), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain including a costimulatory domain and / or a primary signaling domain). In one embodiment, the CAR molecule comprises an antibody or antibody fragment comprising an anti-CD19 binding domain as described herein (e.g., a mouse or humanized antibody or antibody fragment that specifically binds to CD19 as described herein), a transmembrane domain as described herein, and an intracellular signaling domain as described herein (e.g., an intracellular signaling domain including a costimulatory domain and / or a primary signaling domain as described herein).

[0105] In one embodiment, the CAR molecule includes an anti-CD19 binding domain comprising one or more (e.g., all three) of the anti-CD19 binding domains described herein, including one or more (e.g., all three) of the LC CDRs and one or more (e.g., all three) of the anti-CD19 binding domains described herein, including one or more (e.g., all three) of the LC CDRs and one or more (e.g., all three) of the HC CDRs. In one embodiment, the anti-CD19 binding domain comprises one or more (e.g., all three) of the anti-CD19 binding domains described herein, namely, heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3), for example, the anti-CD19 binding domain has two variable heavy chain regions, each containing HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the anti-CD19 binding domain comprises the mouse light chain variable region and / or the mouse heavy chain variable region as described herein (e.g., in Table 3). In one embodiment, the anti-CD19 binding domain is an scFv containing the mouse light chain and mouse heavy chain of the amino acid sequences in Table 3. In one embodiment, the anti-CD19 binding domain (e.g., scFv) includes a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the light chain variable region provided in Table 3 or a sequence having 95-99% identity with the amino acid sequence of Table 3, but with no more than 30, 20, or 10 modifications (e.g., substitutions), and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the heavy chain variable region provided in Table 3 or a sequence having 95-99% identity with the amino acid sequence of Table 3, but with no more than 30, 20, or 10 modifications (e.g., substitutions).In one embodiment, the anti-CD19 binding domain includes the sequence of SEQ ID NO: 59, or a sequence having 95-99% identity thereto. In one embodiment, the anti-CD19 binding domain is an scFv, and a light chain variable region, including the amino acid sequence described herein, for example in Table 3, is attached to a heavy chain variable region, including the amino acid sequence described herein, for example, via a linker, for example, a linker described herein. In one embodiment, the anti-CD19 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, for example 3 or 4 (SEQ ID NO: 53). The light chain variable region and heavy chain variable region of the scFv may be, for example, in the following configuration: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0106] In one embodiment, the CAR molecule includes a humanized anti-CD19 binding domain comprising one or more (e.g., all three) of the light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3) of the humanized anti-CD19 binding domain described herein, and one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, comprising one or more (e.g., all three) of the LC CDR and one or more (e.g., all three) of the HC CDR. 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 humanized anti-CD19 binding domain described herein, such that the humanized anti-CD19 binding domain has two variable heavy chain regions, each containing HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the light chain variable region comprises one, two, three, or all four framework regions of the VK3_L125 germline-derived sequence. In one embodiment, the light chain variable region has modifications (e.g., substitutions, e.g., substitutions of one or more amino acids found at corresponding positions in the mouse light chain variable region of SEQ ID NO: 58, e.g., substitutions at one or more positions 71 and 87). In one embodiment, the heavy chain variable region includes one, two, three, or all four framework regions of the VH4_4-59 germline-derived sequence. In one embodiment, the heavy chain variable region has modifications (e.g., substitutions, e.g., substitutions of one or more amino acids found at corresponding positions in the mouse heavy chain variable region of SEQ ID NO: 58, e.g., substitutions at one or more positions 71, 73, and 78).In one embodiment, the humanized anti-CD19 binding domain includes a light chain variable region as described herein (e.g., in Table 2) and / or a heavy chain variable region as described herein (e.g., in Table 2). In one embodiment, the humanized anti-CD19 binding domain is an scFv including the light and heavy chains of the amino acid sequences in Table 2. In one embodiment, the humanized anti-CD19 binding domain (e.g., scFv) includes a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the light chain variable region provided in Table 2 or a sequence having 95-99% identity with the amino acid sequence of Table 2, but with no more than 30, 20, or 10 modifications (e.g., substitutions), and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the heavy chain variable region provided in Table 2 or a sequence having 95-99% identity with the amino acid sequence of Table 2, but with no more than 30, 20, or 10 modifications (e.g., substitutions). In one embodiment, the humanized anti-CD19 binding domain includes a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a sequence having 95-99% identity thereto. In one embodiment, the humanized anti-CD19 binding domain is an scFv, and a light chain variable region containing an amino acid sequence, for example, as described herein in Table 2, is attached to a heavy chain variable region containing an amino acid sequence, for example, as described herein, via a linker, for example, as 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, for example, 3 or 4 (SEQ ID NO: 53). The light chain variable region and heavy chain variable region of the scFv may be, for example, in the following configuration: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0107] In one embodiment, the CAR molecule includes one or more (e.g., two, three, four, five, or six) anti-CD19 binding domains encompassing LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 from the constructs of Tables 4 and 5, e.g., mouse_CART19, humanized_CART19a, humanized_CART19b, or humanized_CART19c.

[0108] In one embodiment, the CAR molecule comprises a leader sequence, for example, the leader sequence described herein, for example, SEQ ID NO: 13, or a leader sequence having 95-99% identity therewith; and anti-CD19 binding domains described herein, for example, LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and1, and HC CDR1, HC CDR1, HC CDR1, and HC CDR1, HC CDR1, HC CDR1, and The domain includes an anti-CD19 binding domain containing CDR3, e.g., the mouse anti-CD19 binding domain described in Table 3, the humanized anti-CD19 binding domain described in Table 2, or a sequence having 95-99% identity thereto; a hinge region, e.g., a hinge region described herein, e.g., of Sequence ID No. 14, or a hinge region having 95-99% identity thereto; a transmembrane domain, e.g., a transmembrane domain described herein, e.g., a transmembrane domain having the sequence of Sequence ID No. 15, or a sequence having 95-99% identity thereto; and an intracellular signaling domain, e.g., an intracellular signaling domain described herein (e.g., an intracellular signaling domain including a co-stimulatory domain and / or a primary signaling domain). In one embodiment, the intracellular signaling domain includes a 4-1BB co-stimulatory domain having a co-stimulatory domain, for example, the co-stimulatory domain described herein, for example, the sequence of SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence having 95-99% identity thereto; and / or a primary signaling domain, for example, the primary signaling domain described herein, for example, the sequence of SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence having 95-99% identity thereto.

[0109] In one embodiment, the CAR molecule is at least one, two, or three of the amino acid sequences of SEQ ID NO: 58, 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 , an amino acid sequence having 4, 5, 10, 15, 20 or 30 modifications (e.g., substitutions) but 60, 50 or 40 or fewer modifications (e.g., substitutions), or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 58, 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 (e.g., consisting of ).

[0110] The present invention generally relates, in some embodiments, to the use of cells modified to express CARs, such as T cells or natural killer (NK) cells, in combination with one or more B cell inhibitors, to treat diseases associated with the expression of surface antigen classification 19 protein (CD19). In some embodiments, the B cell inhibitor is one or more inhibitors of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0111] In some embodiments, the CD19 inhibitor comprises an antibody molecule, for example, an antibody molecule having a CD19 binding sequence as described herein. For example, the antibody molecule may contain a CDR or VH and VL sequence as described in any of Tables 2, 3, 4, and 5, or a sequence homologous thereto, for example, having 95-99% identity thereto. The antibody molecule may contain a CD19 binding region having a sequence, for example, described in relation to CAR in this section.

[0112] In embodiments, the B cell inhibitor is selected from inhibitory nucleic acids, soluble ligands, antibodies or their antigen-binding fragments, CARs, or CAR-expressing cells that bind to one or more B cell antigens, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0113] CD20 binding domain and results In some embodiments, the Disclosure provides CD20 inhibitors or binding domains, e.g., CD20 inhibitors or binding domains as described herein. The Disclosure also provides nucleic acids encoding CD20 binding domains, e.g., CARs containing CD20 binding domains. The composition may also include a second agent, e.g., anti-CD19 CAR-expressing cells or CD19 binding domains. The agent may be encoded by a single nucleic acid, e.g., or by different nucleic acids.

[0114] In some embodiments, the CD20 inhibitor or binding domain is administered as monotherapy. In some embodiments, the CD20 inhibitor or binding domain is administered in combination with a second agent, such as anti-CD19 CAR-expressing cells.

[0115] CD20 inhibitors may be, for example, small molecules, antibodies or their antigen-binding fragments, CARs, or CAR-expressing cells. In one embodiment, the CD20 inhibitor is an anti-CD20 antibody or a fragment thereof. In one embodiment, the antibody is a monospecific antibody, and in another embodiment, the antibody is a bispecific antibody. In one embodiment, the CD20 inhibitor is a chimeric mouse / human monoclonal antibody, such as rituximab. In one embodiment, the CD20 inhibitor is a human monoclonal antibody such as ofatumumab. In one embodiment, the CD20 inhibitor is a humanized antibody such as ocrelizumab, vertuzumab, obinutuzumab, okalatuzumab, or PRO131921 (Genentech). In one embodiment, the CD20 inhibitor is a fusion protein containing a portion of an anti-CD20 antibody, such as TRU-015 (Trubion Pharmaceuticals).

[0116] In one embodiment, the CD20 inhibitor is anti-CD20 expressing cells, such as CD20 CART or CD20 expressing NK cells.

[0117] In some embodiments, the CD20-CAR comprises an arbitrary leader sequence (e.g., any leader sequence described herein), an extracellular antigen-binding domain, a hinge (e.g., a hinge described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one embodiment, a typical CD20 CAR construct comprises an arbitrary leader sequence (e.g., a leader sequence described herein), an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular co-stimulatory domain (e.g., an intracellular co-stimulatory domain described herein), and an intracellular stimulatory domain.

[0118] In one embodiment, the CD20 binding domain includes one or more (e.g., all three) of the light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3) of the CD20 binding domain described herein, and / or one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the CD20 binding domain described herein, and includes, for example, one or more, e.g., all three, of LC CDRs and one or more, e.g., all three, of HC CDRs. These CDRs may be, for example, those shown in Tables 12A, 12B, and / or Table 13. In one embodiment, the CD20 binding domain includes one or more (e.g., all three) heavy chain complementarity determination regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the CD20 binding domain described herein, for example, the CD20 binding domain has two variable heavy chain regions, each containing HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the CD20 binding domain includes light chain variable regions and / or heavy chain variable regions as described herein (e.g., in Table 15A or 15B). In one embodiment, the CD20 binding domain includes heavy chain variable regions as described herein (e.g., in Table 14A or 14B), for example, at least two heavy chain variable regions as described herein (e.g., in Table 14A or 14B). In one embodiment, the CD20 binding domain is an scFv containing the light and heavy chains of the amino acid sequences shown in Table 14A, 14B, 15A, or 15B.In one embodiment, the CD20-binding domain (e.g., scFv) includes a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the light chain variable region provided in Table 15A or 15B, or a sequence having 95-99% identity with the amino acid sequence of Table 15A or 15B, but with no more than 30, 20, or 10 modifications (e.g., substitutions); and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the heavy chain variable region provided in Table 14A or 14B, or a sequence having 95-99% identity with the amino acid sequence of Table 14A or 14B, but with no more than 30, 20, or 10 modifications (e.g., substitutions). The CD20-binding domain may be, for example, part of an antibody molecule or a CAR molecule.

[0119] In one embodiment, the CAR molecule includes one or more (e.g., two, three, four, five, or six) anti-CD20 binding domains encompassing LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 from the constructs of Tables 12A, 12B, and / or 13, e.g., CAR20-1, CAR20-2, CAR20-3, CAR20-4, CAR20-5, CAR20-6, CAR20-7, CAR20-8, CAR20-9, CAR20-10, CAR20-11, CAR20-12, CAR20-13, CAR20-14, CAR20-15, or CAR20-16.

[0120] In one embodiment, the CAR molecule includes an anti-CD22 binding domain encompassing the VL and / or VH of the constructs of Table 14A or 14B and 15A or 15B, e.g., CAR20-1, CAR20-2, CAR20-3, CAR20-4, CAR20-5, CAR20-6, CAR20-7, CAR20-8, CAR20-9, CAR20-10, CAR20-11, CAR20-12, CAR20-13, CAR20-14, CAR20-15, or CAR20-16.

[0121] Prior to the CD20 scFv, there may be an arbitrary leader sequence, such as that provided in SEQ ID NO: 13, followed by an arbitrary hinge sequence, such as that provided in SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, a transmembrane region, such as that provided in SEQ ID NO: 15, an intracellular signaling domain encompassing SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3 zeta sequence encompassing SEQ ID NO: 17 or SEQ ID NO: 43, for example, the domains being contiguous and within the same reading frame to form a single fusion protein.

[0122] Further embodiments include nucleotide sequences encoding any of the polypeptides in Tables 11A to 15B. Further embodiments include nucleotide sequences encoding any of the polypeptides in Tables 11A to 15B, as well as each of the domains of SEQ ID NOs: 13, 14, 15, 16, 17 and optionally 51.

[0123] In one embodiment, the CD20-binding domain characterizes a specific functional feature or property of the antibody or antibody fragment. For example, in one embodiment, a portion of the CAR composition of the present invention, including an antigen-binding domain, specifically binds to human CD20 or a fragment thereof.

[0124] In one embodiment, the CD20-binding domain is a fragment, e.g., a single-chain variable fragment (scFv). In one embodiment, the CD20-binding domain is Fv, Fab, (Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody [e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)]. In one embodiment, the antibodies and their fragments of the present invention bind to the CD20 protein or its fragment with wild-type or enhanced affinity. In some cases, human scFv may be derived from a display library.

[0125] In one embodiment, the CD20 binding domain, e.g., scFv, includes at least one mutation, such that the mutated scFv confers improved stability to the CART20 construct. In another embodiment, the CD20A binding domain, e.g., scFv, includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations, such that the mutated scFv confers improved stability to the CART20 construct, such that the mutated scFv, e.g., includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations, such as those arising from a humanization process.

[0126] In some embodiments, the CD20 inhibitor comprises an antibody molecule, for example, an antibody molecule having a CD20 binding sequence as described herein. For example, the antibody molecule may contain a CDR or VH and VL sequence listed in any of Tables 11A to 15B, or a sequence homologous thereto, for example, having 95 to 99% identity thereto. The antibody molecule may contain a CD20 binding region having a sequence, for example, one described in relation to CAR in this section.

[0127] In one embodiment, the disclosure provides a population of CAR-expressing cells, such as CART cells, comprising a mixture of cells expressing CD19 CARs and cells expressing CD20 CARs. For example, in one embodiment, the population of CART cells may comprise a first cell expressing CD19 CARs and a second cell expressing CD20 CARs.

[0128] In some embodiments, the binding domain or antibody molecule described herein binds to the same (or substantially the same) or overlapping (or substantially overlapping) epitope as a second antibody molecule against CD20, the second antibody molecule being an antibody molecule described herein, for example, an antibody molecule selected from Tables 11A to 15B. In some embodiments, the binding domain or antibody molecule described herein competes with a second antibody molecule against CD20 for binding to the same (or substantially the same) or overlapping (or substantially overlapping) epitope, and / or binds to such an epitope, the second antibody molecule being an antibody molecule described herein, for example, an antibody molecule selected from Tables 11A to 15B, for example, an antibody molecule determined by the method described in Example 25. In some embodiments, the biparatopic CD20-binding domain binds to a first epitope, for example, an epitope conjugated by an antibody molecule selected from Tables 11A to 15B, and the biparatopic-binding domain also binds to a second epitope, for example, a second epitope conjugated by an antibody molecule selected from Tables 11A to 15B. In some embodiments, the disclosure provides a method of treatment comprising administering a first CD20-binding domain that binds to a first epitope, for example, an epitope conjugated by an antibody molecule selected from Tables 11A to 15B, and a second CD20-binding domain that binds to a second epitope, for example, a second epitope conjugated by an antibody molecule selected from Tables 11A to 15B. In some embodiments, the CD20-binding domain is part of a CAR molecule, for example, expressed by CAR-expressing cells.

[0129] CD22-binding domain and results In some embodiments, the Disclosure provides CD22 inhibitors or binding domains, e.g., CD22 inhibitors or binding domains as described herein. The Disclosure also provides nucleic acids encoding CD22 binding domains, e.g., CARs containing CD22 binding domains. The composition may also contain a second agent, e.g., anti-CD19 CAR-expressing cells or CD19 binding domains. The agent may be encoded by, for example, a single nucleic acid or by different nucleic acids.

[0130] In some embodiments, the CD22 inhibitor or binding domain is administered as monotherapy. In some embodiments, the CD22 inhibitor or binding domain is administered in combination with a second agent, such as anti-CD19 CAR-expressing cells.

[0131] CD22 inhibitors may be, for example, small molecules, antibodies or their antigen-binding fragments, CARs, or CAR-expressing cells. In one embodiment, the CD22 inhibitor is an anti-CD22 antibody or a fragment thereof. In one embodiment, the antibody is a monospecific antibody, and in another embodiment, the antibody is a bispecific antibody. In one embodiment, the antibody is a monospecific antibody that may be conjugated to a second drug, such as a chemotherapeutic agent. For example, in one embodiment, the antibody is an anti-CD22 monoclonal antibody-MMAE conjugate (e.g., DCDT2980S). In one embodiment, the antibody is an scFv of an anti-CD22 antibody, for example, an scFv of antibody RFB4. This scFv may be fused to all or a fragment of Pseudomonas aeruginosa exotoxin-A (e.g., BL22). In one embodiment, the antibody is a humanized anti-CD22 monoclonal antibody (e.g., epratuzumab). In one embodiment, the antibody or fragment comprises an Fv portion of an anti-CD22 antibody, which may be covalently fused to all or a fragment (or a 38kDa fragment) of Pseudomonas aeruginosa exotoxin-A (e.g., moxetumomab pasudotox). In another embodiment, the anti-CD22 antibody is an anti-CD19 / CD22 bispecific antibody, which may be conjugated to the toxin. For example, in one embodiment, the anti-CD22 antibody comprises an anti-CD19 / CD22 bispecific portion (e.g., two scFv ligands that recognize human CD19 and CD22) which may be conjugated to all or part of diphtheria toxin (DT), e.g., diphtheria toxin (DT), DT 390, e.g., ligand-directed toxin, e.g., DT2219ARL, or two scFv ligands that recognize human CD19 and CD22. In another embodiment, the bispecific moiety (e.g., anti-CD19 / anti-CD22) is linked to a toxin such as a deglycosylated lysine A chain (e.g., Combotox).

[0132] In one embodiment, the CD22 inhibitor is anti-CD22 expressing cells, such as CD22 CART or CD22 expressing NK cells.

[0133] In one embodiment, the disclosure provides a population of CAR-expressing cells, e.g., CART cells, comprising a mixture of cells expressing CD19 CARs and cells expressing CD22 CARs. For example, in one embodiment, the population of CART cells may comprise a first group of cells expressing CD19 CARs and a second group of cells expressing CD22 CARs. In another example, the population of CAR T cells may comprise a single population expressing more than one, e.g., two, three, four, five, or six or more CARs, e.g., CD19 CARs and CD22 CARs.

[0134] In some embodiments, the CD22-CAR comprises an arbitrary leader sequence (e.g., any leader sequence described herein), an extracellular antigen-binding domain, a hinge (e.g., a hinge described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one embodiment, a typical CD22-CAR construct comprises an arbitrary leader sequence (e.g., a leader sequence described herein), an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular co-stimulatory domain (e.g., an intracellular co-stimulatory domain described herein), and an intracellular stimulatory domain.

[0135] In one embodiment, the CD22-binding domain includes one or more (e.g., all three) of the light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3) of the CD22-binding domain described herein, and / or one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the CD22-binding domain described herein, and includes, for example, one or more, e.g., all three, of LC CDRs and one or more, e.g., all three, of HC CDRs. These CDRs may be, for example, one or more CDRs from Tables 7A, 7B, 7C, 8A, and / or Table 8B. In one embodiment, the CD22-binding domain includes one or more (e.g., all three) of the CD22-binding domains described herein, such that the CD22-binding domain has two variable heavy chain regions, each containing HC CDR1, HC CDR2, and HC CDR3, respectively. In one embodiment, the CD22-binding domain includes a light chain variable region described herein (e.g., in Table 10A or 10B) and / or a heavy chain variable region described herein (e.g., in Table 9A or 9B). In one embodiment, the CD22-binding domain includes a heavy chain variable region described herein (e.g., in Table 9A or 9B), such as at least two heavy chain variable regions described herein (e.g., in Table 9A or 9B). In one embodiment, the CD22-binding domain is an scFv containing the light and heavy chains of the amino acid sequences shown in Table 9A or 9B and 10A or 10B.In one embodiment, the CD22-binding domain (e.g., scFv) includes a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the light chain variable region provided in Table 10A or 10B, or a sequence having 95-99% identity with the amino acid sequence of Table 10A or 10B, but with no more than 30, 20, or 10 modifications (e.g., substitutions); and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the heavy chain variable region provided in Table 9A or 9B, or a sequence having 95-99% identity with the amino acid sequence of Table 9A or 9B, but with no more than 30, 20, or 10 modifications (e.g., substitutions). The CD22-binding domain may be, for example, part of an antibody molecule or a CAR molecule.

[0136] In one embodiment, the CAR molecule is a construct of Table 7A, 7B, 7C, 8A, and / or 8B, e.g., m971, CAR22-1, CAR22-2, CAR22-3, CAR22-4, CAR22-5, CAR22-6, CAR22-7, CAR22-8, CAR22-9, CAR22-10, CAR22-11, CAR22-12, CAR22-13, CAR22-14, CAR22-15, CAR22-16, CAR22-17, CAR22-18, CAR2 2-19, CAR22-20, CAR22-21, CAR22-22, CAR22-23, CAR22-24, CAR22-25, CAR22-26, CAR22-27, CAR22-28, CAR22-29, CAR22-30, CAR22-31, CAR22-32, CAR22-33, CAR22-34, CAR22-35, CAR22-36, CAR22-37, or CAR22-38 contain one or more (e.g., two, three, four, five, or six) anti-CD22 binding domains that include LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3.

[0137] In one embodiment, the CAR molecule is a construct of Table 9A, 9B, 10A and / or 10B, for example, m971, CAR22-1, CAR22-2, CAR22-3, CAR22-4, CAR22-5, CAR22-6, CAR22-7, CAR22-8, CAR22-9, CAR22-10, CAR22-11, CAR22-12, CAR22-13, CAR22-14, CAR22-15, CAR22-16, CAR22-17, CAR22-18, CAR22-19, CAR22-2 Contains an anti-CD22 binding domain encompassing VL and / or VH of sequences 0, CAR22-21, CAR22-22, CAR22-23, CAR22-24, CAR22-25, CAR22-26, CAR22-27, CAR22-28, CAR22-29, CAR22-30, CAR22-31, CAR22-32, CAR22-33, CAR22-34, CAR22-35, CAR22-36, CAR22-37, or CAR22-38, or sequences having 95-99% identity with them.

[0138] Prior to the scFv, there may be an arbitrary leader sequence, such as the one provided in SEQ ID NO: 13, followed by an arbitrary hinge sequence, such as the one provided in SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, a transmembrane region, such as the one provided in SEQ ID NO: 15, an intracellular signaling domain encompassing SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3 zeta sequence encompassing SEQ ID NO: 17 or SEQ ID NO: 43, for example, the domains being contiguous and within the same reading frame to form a single fusion protein.

[0139] Further embodiments include nucleotide sequences encoding any of the polypeptides in Tables 6A to 10B. Further embodiments include nucleotide sequences encoding any of the polypeptides in Tables 6A to 10B, as well as each of the domains of SEQ ID NOs: 13, 14, 15, 16, 17 and optionally 51.

[0140] In one embodiment, the CD22 binding domain is characterized by certain functional features or properties of an antibody or antibody fragment. For example, in one embodiment, a portion of the CAR composition of the invention comprising an antigen binding domain specifically binds to human CD22 or a fragment thereof.

[0141] In one embodiment, the CD22 binding domain is a fragment, such as a single-chain variable fragment (scFv). In one embodiment, the CD22 binding domain is an Fv, Fab, (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 the CD22 protein or a fragment thereof with wild-type or enhanced affinity. In some cases, the human scFv may be derived from a display library.

[0142] In one embodiment, the CD22 binding domain, such as an scFv, comprises at least one mutation such that the mutated scFv confers improved stability to the CART-22 construct. In another embodiment, the CD22 binding domain, such as an scFv, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations resulting, for example, from a humanization process such that the mutated scFv confers improved stability to the CART-22 construct.

[0143] In some embodiments, the CD22 inhibitor comprises an antibody molecule, such as an antibody molecule having a CD22 binding sequence as described herein. For example, the antibody molecule may comprise a CDR or VH and VL described in any of Tables 6A - 10B, or a sequence having homology thereto, such as 95 - 99% identity thereto. The antibody molecule may comprise a CD22 binding region having a sequence described, for example, in relation to CARs in this section.

[0144] In one embodiment, the present disclosure provides a population of CAR-expressing cells, such as CAR T cells, comprising a mixture of cells expressing a CD19 CAR and cells expressing a CD22 CAR. For example, in one embodiment, the population of CAR T cells can include a first cell expressing a CD19 CAR and a second cell expressing a CD22 CAR.

[0145] In some aspects, the binding domains or antibody molecules described herein bind to the same (or substantially the same) or overlapping (or substantially overlapping) epitopes as a second antibody molecule against CD22, and the second antibody molecule is an antibody molecule described herein, such as an antibody molecule selected from Tables 6A-10B. In some embodiments, the binding domains or antibody molecules described herein compete for binding to the same (or substantially the same) or overlapping (or substantially overlapping) epitopes as a second antibody molecule against CD22, and / or bind to such epitopes, and the second antibody molecule is an antibody molecule described herein, such as an antibody molecule selected from Tables 6A-10B, such as an antibody molecule as determined by the method described in Example 25. In some embodiments, the bispecific CD22 binding domain binds to a first epitope, such as an epitope bound by an antibody molecule selected from Tables 6A-10B, and the bispecific binding domain also binds to a second epitope, such as a second epitope bound by an antibody molecule selected from Tables 6A-10B. In some aspects, the present disclosure provides a method of treatment comprising administering a first CD22 binding domain that binds to a first epitope, such as an epitope bound by an antibody molecule selected from Tables 6A-10B, and a second CD22 binding domain that binds to a second epitope, such as a second epitope bound by an antibody molecule selected from Tables 6A-10B. In some embodiments, the CD22 binding domain is part of a CAR molecule, such as expressed by a CAR-expressing cell.

[0146] In some embodiments, the CD22 binding domain binds to one or more Ig-like domains 1, 2, 3, 4, 5, 6, or 7 of CD22. In some embodiments, the CD22 binding domain binds to domains 1 and 2, or to domains 3 and 4, or to domains 5, 6, and 7.

[0147] In some embodiments, the Disclosure provides a method for treating CD19-negative cancers, such as leukemia, such as ALL, such as B-ALL, comprising administering a CD22 inhibitor, such as CD22-binding domain or CD22 CAR-expressing cells as described herein. In some embodiments, the method includes the step of determining whether the cancer is CD19-negative. In some embodiments, the subject has previously received a CD19 inhibitor, such as CD19 CAR-expressing cells, and is resistant to the CD19 inhibitor, has relapsed, or is ineffective against the CD19 inhibitor.

[0148] ROR1 inhibitors The ROR1 inhibitor may be, for example, a small molecule, an antibody, or a fragment thereof. In one embodiment, the ROR1 inhibitor is an anti-ROR1 antibody or a fragment thereof. In one embodiment, the anti-ROR1 antibody or a fragment thereof is a monoclonal antibody, such as cirmtuzumab.

[0149] In one embodiment, the ROR1 inhibitor is anti-ROR1 expressing cells, such as ROR1 CART or ROR1 expressing NK cells.

[0150] In some embodiments, the ROR1-CAR comprises an arbitrary leader sequence (e.g., any leader sequence described herein), an extracellular antigen-binding domain, a hinge (e.g., a hinge described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one embodiment, a typical ROR1-CAR construct comprises an arbitrary leader sequence (e.g., a leader sequence described herein), an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular co-stimulatory domain (e.g., an intracellular co-stimulatory domain described herein), and an intracellular stimulatory domain.

[0151] In one embodiment, the ROR1 binding domain includes an scFv portion, for example, a human scFv portion. Prior to the scFv, there may be an arbitrary reader sequence, such as that provided in SEQ ID NO: 13, followed by an arbitrary hinge sequence, such as that provided in SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, a transmembrane region, such as that provided in SEQ ID NO: 15, an intracellular signaling domain encompassing SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3 zeta sequence encompassing SEQ ID NO: 17 or SEQ ID NO: 43, for example, these domains being contiguous and within the same reading frame to form a single fusion protein.

[0152] In some embodiments, the disclosure encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a ROR1 CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a ROR1 binding domain, for example, a ROR1 binding domain that is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain, as described herein. Typical intracellular signaling domains that can be used in a CAR include, but are not limited to, one or more intracellular signaling domains of the same kind, such as CD3-zeta, CD28, 4-1BB, and similar domains. In some cases, the CAR may comprise any combination of CD3-zeta, CD28, 4-1BB, and similar domains.

[0153] In one embodiment, the ROR1 binding domain characterizes a specific functional feature or property of the antibody or antibody fragment. For example, in one embodiment, a portion of the CAR composition of the present invention, including the antigen-binding domain, specifically binds to human ROR1 or a fragment thereof. In a particular embodiment, the scFv is contiguous with the leader sequence and resides within the same reading frame. In one embodiment, the leader sequence is a polypeptide sequence provided as SEQ ID NO: 13.

[0154] In one embodiment, the ROR1-binding domain is a fragment, e.g., a single-chain variable fragment (scFv). In one embodiment, the ROR1-binding domain is Fv, Fab, (Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody [e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)]. In one embodiment, the antibodies and their fragments of the present invention bind to the ROR1 protein or its fragments with wild-type or enhanced affinity. In some cases, human scFv may be derived from a display library.

[0155] In one embodiment, the ROR1 binding domain, e.g., scFv, includes at least one mutation, such that the mutated scFv confers improved stability to the ROR1 CART construct. In another embodiment, the ROR1 binding domain, e.g., scFv, includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations resulting from a humanization process, such that the mutated scFv confers improved stability to the ROR1 CART construct.

[0156] In one embodiment, the disclosure provides a population of CAR-expressing cells, such as CART cells, comprising a mixture of cells expressing CD19 CAR and cells expressing ROR1 CAR. For example, in one embodiment, the population of CART cells may comprise a first cell expressing CD19 CAR and a second cell expressing ROR1 CAR.

[0157] CD123 inhibitors CD123 inhibitors may be, for example, small molecules, antibodies or fragments thereof (e.g., monospecific or bispecific antibodies, or fragments thereof); recombinant proteins that bind to CD123, such as fusion proteins; inhibitory nucleic acids; or cells expressing CD123 CAR, such as CD123 CART.

[0158] In one embodiment, the CD123 inhibitor is a recombinant protein containing, for example, a native ligand (or fragment) of the CD123 receptor, such as SL-401 [also known as DT388IL3; University of Texas Southwestern Medical Center].

[0159] In another embodiment, the CD123 inhibitor is an anti-CD123 antibody or a fragment thereof, for example, a monoclonal antibody (e.g., a monospecific or bispecific antibody, or a fragment thereof), such as CSL360 (CSL Limited), CSL 362 (CSL Limited), or MGD006 (MacroGenics).

[0160] In one embodiment, the CD123 inhibitor is anti-CD123 CAR-expressing cells, such as CD123 CART or CD123 CAR-expressing NK cells.

[0161] In some embodiments, the CD123-CAR comprises an arbitrary leader sequence (e.g., any leader sequence described herein), an extracellular antigen-binding domain, a hinge (e.g., a hinge described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one embodiment, a typical CD123-CAR construct comprises an arbitrary leader sequence (e.g., a leader sequence described herein), an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular co-stimulatory domain (e.g., an intracellular co-stimulatory domain described herein), and an intracellular stimulatory domain.

[0162] In one embodiment, the CD123-binding domain includes one or more (e.g., all three) light chain complementarity determination regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the CD20-binding domain described herein, and / or one or more (e.g., all three) heavy chain complementarity determination regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the CD123-binding domain described herein, and includes, for example, one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. These CDRs may be, for example, any of those in Tables 17, 18, 26, or 27. In one embodiment, the CD123-binding domain includes one or more (e.g., all three) of the CD123-binding domains described herein, such that the CD123-binding domain has two variable heavy chain regions, each containing the HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the CD123-binding domain includes the light chain variable region and / or the heavy chain variable region described herein. In one embodiment, the CD123-binding domain includes the heavy chain variable region described herein, for example, at least two heavy chain variable regions described herein. In one embodiment, the CD123-binding domain is an scFv containing the light and heavy chains of the amino acid sequences in Table 16 or 25.In one embodiment, the CD123 binding domain (e.g., scFv) includes a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the light chain variable region in Table 16 or 25 or a sequence having 95-99% identity with the light chain variable region in Table 16 or 25, but with no more than 30, 20, or 10 modifications (e.g., substitutions), and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the heavy chain variable region in Table 16 or 25 or a sequence having 95-99% identity with the heavy chain variable region in Table 16 or 25, but with no more than 30, 20, or 10 modifications (e.g., substitutions).

[0163] In one embodiment, the CAR molecule includes one or more (e.g., two, three, four, five, or six) anti-CD123 binding domains encompassing LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 from the constructs in Tables 17 and 18, e.g., CAR123-1, CAR123-2, CAR123-3, or CAR123-4. In one embodiment, the CAR molecule includes one or more (e.g., two, three, four, five, or six) anti-CD123 binding domains encompassing LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 from the constructs in Tables 26 and 27, e.g., hzCAR123.

[0164] Prior to the CD123 scFv, there may be any leader sequence, such as that provided in SEQ ID NO: 13, followed by any hinge sequence, such as that provided in SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, any transmembrane region, such as that provided in SEQ ID NO: 15, an intracellular signaling domain encompassing SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3 zeta sequence encompassing SEQ ID NO: 17 or SEQ ID NO: 43, for example, the domains being contiguous and within the same reading frame to form a single fusion protein.

[0165] Further embodiments include nucleotide sequences encoding any of the polypeptides of Tables 16-27. Further embodiments include nucleotide sequences encoding any of the polypeptides of Tables 16-27 and each of the domains of SEQ ID NOs: 13, 14, 15, 16, 17 and optionally 51.

[0166] In one embodiment, the CD123 binding domain is characterized by certain functional features or properties of an antibody or antibody fragment. For example, in one embodiment, a portion of the CAR composition of the invention comprising an antigen binding domain specifically binds to human CD123 or a fragment thereof.

[0167] In one embodiment, the CD123 binding domain is a fragment, such as a single-chain variable fragment (scFv). In one embodiment, the CD123 binding domain is an Fv, Fab, (Fab’)2, or bifunctional (e.g., bispecific) hybrid antibody [e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)]. In one aspect, the antibodies and fragments thereof of the invention bind to the CD123 protein or a fragment thereof with wild-type or enhanced affinity. In some cases, the human scFv may be derived from a display library.

[0168] In one embodiment, the CD123 binding domain, such as an scFv, comprises at least one mutation so that the mutated scFv confers improved stability to the CART123 construct. In another embodiment, the CD123 binding domain, such as an scFv, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations resulting, for example, from a humanization process so that the mutated scFv confers improved stability to the CART1十二条3 construct.

[0169] In some embodiments, the CD123 inhibitor comprises an antibody molecule, for example, an antibody molecule having a CD123 binding sequence as described herein. For example, the antibody molecule may contain a CDR or VH and VL sequence listed in any of Tables 16 to 27, or a sequence homologous thereto, for example, having 95 to 99% identity thereto. The antibody molecule may contain a CD123 binding region having a sequence, for example, described in relation to CAR in this section.

[0170] In one embodiment, the disclosure provides a population of CAR-expressing cells, such as CART cells, comprising a mixture of cells expressing CD19 CARs and cells expressing CD123 CARs. For example, in one embodiment, the population of CART cells may comprise a first cell expressing CD19 CARs and a second cell expressing CD123 CARs.

[0171] CD10 inhibitors CD10 inhibitors may be, for example, small molecules, antibodies or their fragments (e.g., monospecific or bispecific antibodies, or their fragments); recombinant proteins that bind to CD10, such as fusion proteins; inhibitory nucleic acids; or cells expressing CD10 CAR, such as CD10 CART.

[0172] In one embodiment, the CD10 inhibitor includes small molecules such as sacubitril (Novartis), valsartan / sacubitril (Novartis), omapatrilate (Bristol-Myers Squibb), RB-101, UK-414,495 (Pfizer), or pharmaceutically acceptable salts or derivatives thereof.

[0173] In one embodiment, the CD10 inhibitor is an anti-CD10 CAR-expressing cell, such as a CD10 CART or CD10 CAR-expressing NK cell.

[0174] In one embodiment, the disclosure provides a population of CAR-expressing cells, such as CART cells, comprising a mixture of cells expressing CD19 CARs and cells expressing CD10 CARs. For example, in one embodiment, the population of CART cells may comprise a first cell expressing CD19 CARs and a second cell expressing CD10 CARs.

[0175] CD34 inhibitor CD34 inhibitors may be, for example, small molecules, antibodies or fragments thereof (e.g., monospecific or bispecific antibodies, or fragments thereof); recombinant proteins that bind to CD34, such as fusion proteins; inhibitory nucleic acids; or cells expressing CD34 CAR, such as CD34 CART.

[0176] In one embodiment, the CD34 inhibitor comprises a monoclonal antibody or fragment thereof that targets CD34, or an immunoliposome containing an anti-CD34 monoclonal antibody or fragment thereof.

[0177] In one embodiment, the CD34 inhibitor is anti-CD34 CAR-expressing cells, such as CD34 CART or CD34 CAR-expressing NK cells.

[0178] In one embodiment, the disclosure provides a population of CAR-expressing cells, such as CART cells, comprising a mixture of cells expressing CD19 CARs and cells expressing CD34 CARs. For example, in one embodiment, the population of CART cells may comprise a first cell expressing CD19 CARs and a second cell expressing CD34 CARs.

[0179] FLT-3 inhibitors FLT-3 inhibitors may be, for example, small molecules, antibodies or fragments thereof (e.g., monospecific or bispecific antibodies, or fragments thereof); recombinant proteins that bind to FLT-3, such as fusion proteins; inhibitory nucleic acids; or cells expressing FLT-3 CAR, such as FLT-3 CART.

[0180] In some embodiments, the FLT-3 inhibitor includes small molecules such as quizartinib (Ambit Biosciences), midostaurin (Technische Universitat Dresden), sorafenib (Bayer and Onyx Pharmaceuticals), sunitinib (Pfizer), restaurtinib (Cephalon), or pharmaceutically acceptable salts or derivatives thereof.

[0181] In one embodiment, the FLT-3 inhibitor is an anti-FLT-3 CAR-expressing cell, such as FLT-3 CART or FLT-3 CAR-expressing NK cell.

[0182] In one embodiment, the disclosure provides a population of CAR-expressing cells, such as CART cells, comprising a mixture of cells expressing CD19 CAR and cells expressing FLT-3 CAR. For example, in one embodiment, the population of CART cells may comprise a first cell expressing CD19 CAR and a second cell expressing FLT-3 CAR.

[0183] CD79b inhibitors In certain embodiments, CD19 CAR-expressing cells are administered together with a CD79b inhibitor. The CD79b inhibitor may be, for example, a small molecule, an antibody or fragment thereof (e.g., a monospecific or bispecific antibody, or a fragment thereof); a recombinant protein that binds to CD79b, such as a fusion protein; an inhibitory nucleic acid; or cells expressing CD79b CAR, such as CD79b CAR-expressing T cells or NK cells. In one embodiment, the CD79b inhibitor is anti-CD79b CAR-expressing cells, such as CD79b CART or CD79b CAR-expressing NK cells. Typical CD79b inhibitors are described in more detail below.

[0184] In one embodiment, the disclosure provides a population of CAR-expressing cells, such as CART cells or CAR-expressing NK cells, comprising a mixture of cells expressing CD19 CARs and cells expressing CD79b CARs. For example, in one embodiment, the population of CAR-expressing cells comprises a first cell expressing CD19 CARs and a second cell expressing CD79b CARs.

[0185] CD179b inhibitors In certain embodiments, CD19 CAR-expressing cells are administered together with a CD179b inhibitor. The CD179b inhibitor may be, for example, a small molecule, an antibody or fragment thereof (e.g., a monospecific or bispecific antibody, or a fragment thereof); a recombinant protein that binds to CD179b, such as a fusion protein; an inhibitory nucleic acid; or cells expressing CD179b CAR, such as CD179b CAR-expressing T cells or NK cells. In one embodiment, the CD79b inhibitor is anti-CD179b CAR-expressing cells, such as CD179b CART or CD179b CAR-expressing NK cells. Typical CD179b inhibitors are described in more detail below.

[0186] In one embodiment, the disclosure provides a population of CAR-expressing cells, such as CART cells or CAR-expressing NK cells, comprising a mixture of cells expressing CD19 CARs and cells expressing CD179b CARs. For example, in one embodiment, the population of CAR-expressing cells comprises a first cell expressing CD20 CARs and a second cell expressing CD179b CARs.

[0187] CD79a inhibitors In certain embodiments, CD19 CAR-expressing cells are administered together with a CD79a inhibitor. The CD79a inhibitor may be, for example, a small molecule, an antibody or fragment thereof (e.g., a monospecific or bispecific antibody, or a fragment thereof); a recombinant protein that binds to CD79a, such as a fusion protein; an inhibitory nucleic acid; or cells expressing CD79a CAR, such as CD79a CAR-expressing T cells or NK cells. In one embodiment, the CD79a inhibitor is anti-CD79a CAR-expressing cells, such as CD79a CART or CD79a CAR-expressing NK cells. Typical CD79a inhibitors are described in more detail below.

[0188] In one embodiment, the disclosure provides a population of CAR-expressing cells, such as CART cells or CAR-expressing NK cells, comprising a mixture of cells expressing CD19 CARs and cells expressing CD79a CARs. For example, in one embodiment, the population of CAR-expressing cells comprises a first cell expressing CD19 CARs and a second cell expressing CD79a CARs.

[0189] CAR molecule The binding domains described herein (for example, binding domains to one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a) may further comprise one or more additional amino acid sequences.

[0190] In one embodiment, the CAR molecule includes a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a 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 includes the sequence of SEQ ID NO: 15. In one embodiment, the transmembrane domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but 20, 10, or 5 or fewer modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 15, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 15.

[0191] In one embodiment, the 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 includes sequence number 14 or sequence number 45, or a sequence having 95-99% identity with them.

[0192] In one embodiment, the CAR molecule further comprises a co-stimulatory domain, for example, a sequence encoding a co-stimulatory domain as described herein. In one embodiment, 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), ICOS (CD278), and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO: 16. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO: 51. In one embodiment, the co-stimulatory domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 20, 10, or 5 modifications (e.g., substitutions), of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51.In one embodiment, the co-stimulatory domain is an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activating molecule (SLAM protein), an activated NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B 7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, N KG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD22) 9) The protein comprises a functional signaling domain selected from the group consisting of ligands that specifically bind to CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83. In embodiments, the co-stimulatory domain comprises 4-1BB, CD27, CD28, or ICOS.

[0193] In one embodiment, the CAR molecule further comprises a sequence encoding an intracellular signaling domain, for example, an intracellular signaling domain as 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 of SEQ ID NO: 16 and / or the sequence of SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and / or the sequence of 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 of SEQ ID NO: 51 and / or the sequence of SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 51 and / or the sequence of SEQ ID NO: 43. In one embodiment, the intracellular signaling domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but 20, 10, or 5 or fewer modifications (e.g., substitutions), of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43. In one embodiment, the intracellular signaling domain includes 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, and the sequences constituting the intracellular signaling domain are expressed as a single polypeptide chain within the same frame.

[0194] In one embodiment, the CAR molecule further comprises a leader sequence, for example, a leader sequence described herein. In one embodiment, the leader sequence comprises the amino acid sequence of SEQ ID NO: 13, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 13.

[0195] In one embodiment, a CAR (e.g., CD19 CAR, ROR1 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, or CD79a CAR) comprises an arbitrary leader sequence (e.g., any leader sequence described herein), an extracellular antigen-binding domain, a hinge (e.g., a hinge described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one embodiment, an exemplary CAR construct comprises an arbitrary leader sequence (e.g., any leader sequence described herein), an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular co-stimulatory domain (e.g., an intracellular co-stimulatory domain described herein), and an intracellular stimulatory domain.

[0196] bispecific antibody A bispecific antibody molecule (which can be administered, for example, alone or as part of a CAR) may contain two VH regions and two VL regions. In some embodiments, the upstream antibody or a portion thereof (e.g., scFv) is arranged such that its VH(VH1) is upstream of its VL(VL1) so that the overall bispecific antibody molecule has a VH1-VL1-VL2-VH2 arrangement, and the downstream antibody or a portion thereof (e.g., scFv) is arranged such that its VL(VL2) is upstream of its VH(VH2). In other embodiments, the upstream antibody or a portion thereof (e.g., scFv) is arranged such that its VL(VL1) is upstream of its VH(VH1) so that the overall bispecific antibody molecule has a VL1-VH1-VH2-VL2 arrangement, and the downstream antibody or a portion thereof (e.g., scFv) is arranged such that its VH(VH2) is upstream of its VL(VL2).

[0197] Dual-specificity CD22 / CD19 inhibitors In one embodiment, the B cell inhibitor comprises a bispecific CAR19 / CAR22 antibody molecule. For example, in some embodiments, the B cell inhibitor comprises one or more amino acid sequences from Table 28, or sequences having 95-99% identity thereto. Further examples of nucleic acids, or sequences having 95-99% identity thereto, according to Table 28 are provided. In one embodiment, the B cell inhibitor comprises a CD19-specific antibody molecule (or a sequence having 95-99% identity thereto) from Table 2 or 3 and a CD22-specific antibody molecule (or a sequence having 95-99% identity thereto) from Table 6A or 6B. In one embodiment, the B cell inhibitor comprises a CD19-specific antibody molecule having one or more CDRs (or sequences with one, two, three, four, five, or six modifications, e.g., substitutions) from Table 4 or 5, and a CD22-specific antibody molecule having a CDR (or sequences with one, two, three, four, five, or six modifications, e.g., substitutions) from Table 7A, 7B, 7C, 8A, or 8B.

[0198] mTOR inhibitors In one embodiment, cells expressing CAR molecules, such as CD19 CAR molecules, CD20 CAR molecules, or CD22 CAR molecules, such as the CAR molecules described herein, which are administered in combination with a B cell inhibitor as appropriate, are co-administered with a low dose of an immune-enhancing mTOR inhibitor. While we do not wish to be bound by theory, it is thought that treatment with a low dose of immune-enhancing drugs (e.g., a dose insufficient to completely suppress the immune system but sufficient to improve immune function) would result in a decrease in PD-1-positive T cells or an increase in PD-1-negative cells. PD-1-positive T cells, rather than PD-1-negative T cells, can be eliminated by contact with cells expressing PD-1 ligands, such as PD-L1 or PD-L2.

[0199] In one embodiment, this approach can be used to optimize the performance of CAR cells described herein in a subject. While we do not wish to be bound by theory, in one embodiment, it is thought that the performance of endogenous, unmodified immune effector cells, such as T cells, will be improved. While we do not wish to be bound by theory, in one embodiment, it is thought that the performance of cells expressing CARs will be improved. In other embodiments, cells modified to express CARs, or expected to be modified, such as T cells, may be treated ex vivo by contacting them with an amount of mTOR inhibitor that increases the number of PD1-negative immune effector cells, such as T cells, or increases the ratio of PD1-negative immune effector cells, such as T cells, to PD1-positive immune effector cells, such as T cells.

[0200] In one embodiment, administration of a low dose of an immune-enhancing mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, or a catalytic inhibitor, is initiated before administration of cells expressing the CAR described herein, such as T cells. In one embodiment, the CAR cells are administered after a sufficient amount of time or sufficient administration of the mTOR inhibitor so that the level of PD1-negative immune effector cells, such as T cells, or the ratio of PD1-negative immune effector cells, such as T cells, to PD1-positive immune effector cells, such as T cells, increases at least temporarily.

[0201] In one embodiment, cells processed to express CAR, such as T cells, are harvested after a sufficient amount of time, or after administration of a low dose of an mTOR inhibitor that sufficiently enhances immunity, so that the level of PD1-negative immune effector cells, such as T cells, or the ratio of PD1-negative immune effector cells, such as T cells, to PD1-positive immune effector cells, such as T cells, in or harvested from a subject increases at least transiently.

[0202] Further features or embodiments of the compositions or methods described herein include one or more of the following:

[0203] In one embodiment, the B cell inhibitor comprises one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1. In another embodiment, the B cell inhibitor comprises an effective number of one or more cells expressing a CAR molecule that binds to one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1.

[0204] In one embodiment, one or more cells expressing a CD19-binding CAR molecule are administered simultaneously with, or before or after, one or more B-cell inhibitors.

[0205] In the embodiment, a subject is identified as having, or possessing, a difference between a level determined by comparing it to the reference level of one or more markers listed in Table 29 in a biological sample, for example, a statistically significant difference.

[0206] In the embodiment, a subject is identified in a biological sample as having, or possessing, a difference between a determined feature and a reference feature with respect to CD19 characteristics, such as mutations that cause frameshifts or immature stop codons or both.

[0207] In the embodiment, subjects are identified as having, or possessing, a difference between a level determined by comparing it to a reference level of Treg cells in a biological sample, for example, a statistically significant difference.

[0208] In one embodiment, the method involves the subject being (i) the level or activity of one or more markers listed in Table 29, (ii) a characteristic of CD19, such as a mutation, such as a mutation that causes a frameshift or immature stop codon or both, or (iii) T in a biological sample REGIf, with respect to one or more levels of cells, a difference is identified between a level determined in comparison to a reference level or a feature determined in comparison to a reference feature, for example, a statistically significant difference, the method includes administering to the subject a therapeutically effective dose of chimeric antigen receptor (CAR) therapy, such as CAR therapy as described herein, e.g., therapy comprising CD19 CAR-expressing cells and, as appropriate, one or more B-cell inhibitors. In one embodiment, the method is to determine that the subject has (i) levels of one or more markers listed in Table 29, (ii) CD19 features, e.g., mutations, e.g., mutations causing frameshift or immature stop codons or both, or (iii) T in a biological sample REG The method comprises determining whether there is a statistically significant difference, for example, between one or more levels or activities of cells and a level determined in comparison to a reference level, or between a level determined in comparison to a reference feature, and administering to the subject a therapeutically effective dose of a chimeric antigen receptor (CAR) therapy, such as a CAR therapy as described herein, such as a therapy comprising CD19 CAR-expressing cells and, as appropriate, one or more B-cell inhibitors. In one embodiment, the method is to determine whether the subject has (i) levels of one or more markers listed in Table 29, (ii) CD19 features, such as mutations, such as mutations causing frameshifts or immature stop codons or both, or (iii) T in a biological sample REGThe method comprises determining whether there is a statistically significant difference, for example, between one or more levels or activities of cells and a level determined in comparison to a reference level, or between a level determined in comparison to a reference feature, and administering to the subject a therapeutically effective dose of a chimeric antigen receptor (CAR) therapy, such as a CAR therapy as described herein, such as a therapy comprising CD19 CAR-expressing cells, and optionally one or more B-cell inhibitors. In one embodiment, the method is to administer to the subject a therapeutically effective dose of a chimeric antigen receptor (CAR) therapy, such as a CAR therapy as described herein, such as a therapy comprising CD19 CAR-expressing cells; and determining whether the subject has (i) levels of one or more markers listed in Table 29, (ii) characteristics of CD19, such as mutations, such as mutations causing frameshifts or immature stop codons or both, or (iii) T in a biological sample REG The process involves determining whether there is a difference, for example, a statistically significant difference, between one or more levels or activities of cells and a level determined in comparison to a reference level, or between a level determined in comparison to a reference feature; and, if a difference exists, administering a therapeutically effective dose of one or more B-cell inhibitors to the target.

[0209] In the embodiment, subjects are identified as having, or possessing, an increase between a determined level and a reference level of Treg cells in a biological sample, for example, a statistically significant increase.

[0210] In the embodiment, subjects are identified as those that have relapsed or are relapsed after treatment with one or more cells expressing a CD19-binding CAR molecule, such as a CD19 CAR.

[0211] In an embodiment, the B cell inhibitor comprises an effective number of one or more cells expressing a CD10-binding CAR molecule, e.g., a CD10 CAR as described herein; a CD20-binding CAR molecule, e.g., a CD20 CAR as described herein; a CD22-binding CAR molecule, e.g., a CD22 CAR as described herein; a CD34-binding CAR molecule, e.g., a CD34 CAR as described herein; a CD123-binding CAR molecule, e.g., a CD123 CAR as described herein; a FLT-3-binding CAR molecule, e.g., a FLT-3 CAR as described herein; or a ROR1-binding CAR molecule, e.g., a ROR1 CAR as described herein.

[0212] In embodiments, the CD19 inhibitor comprises an antibody or antibody fragment comprising a CD19-binding domain, a transmembrane domain, and an intracellular signaling domain including a stimulating domain, wherein the CD19-binding domain comprises one or more (e.g., all three) light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD19 light chain-binding domain amino acid sequence listed in Table 2 or 3, and one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD19 heavy chain-binding domain amino acid sequence listed in Table 2 or 3.

[0213] In the embodiment, the CD19 CAR includes the light chain variable region listed in Table 2 or 3, and one of the heavy chain variable regions listed in Table 2 or 3.

[0214] In an embodiment, the CD19 inhibitor includes a CD19-binding domain comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a sequence having 95-99% identity with them. In an embodiment, the CD19 CAR includes the polypeptide of SEQ ID NO: 58.

[0215] In embodiments, the B cell inhibitor comprises a CD20 CAR, the CD20 CAR comprising an antibody or antibody fragment comprising a CD20-binding domain, a transmembrane domain, and an intracellular signaling domain including a stimulating domain, wherein the CD20-binding domain comprises one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD20 light chain-binding domain amino acid sequence listed in Table 13, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD20 heavy chain-binding domain amino acid sequence listed in Table 12A or 12B.

[0216] In embodiments, the B cell inhibitor comprises a CD22 CAR, the CD22 CAR comprising an antibody or antibody fragment comprising a CD22-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulating domain, wherein the CD22-binding domain comprises one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD22 light chain-binding domain amino acid sequence listed in Tables 8A, 8B, 10A, and / or 10B, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD22 heavy chain-binding domain amino acid sequence listed in Tables 7A, 7B, 7C, 9A, and / or 9B.

[0217] In the embodiment, the CD22 CAR includes one of the light chain variable regions listed in Table 10A or 10B. In the embodiment, the CD22 CAR includes one of the heavy chain variable regions listed in Table 9A or 9B. In the embodiment, the CD22 CAR includes one of the light chain variable regions listed in Table 10A or 10B, and one of the heavy chain variable regions listed in Table 9A or 9B.

[0218] In embodiments, the B cell inhibitor comprises a CAR, the CAR comprising an antibody or antibody fragment comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulating domain, wherein the antigen-binding domain comprises one or more (e.g., all) of light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3), and one or more (e.g., all) of heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3).

[0219] In an embodiment, the B cell inhibitor comprises a CAR containing scFv. In an embodiment, the B cell inhibitor comprises a CAR containing a transmembrane domain, the transmembrane domain comprising a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In an embodiment, the antigen-binding domain is connected to the transmembrane domain by a hinge region. In an embodiment, the hinge region comprises sequence number 14, or a sequence having 95-99% identity thereto. In embodiments, the co-stimulatory domain is a 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 embodiments, the co-stimulatory domain includes MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7- H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITG AD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D , NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), C The functional signaling domain is obtained from a protein selected from the group consisting of ligands that specifically bind to D160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83. In embodiments, the co-stimulatory domain includes the sequence of SEQ ID NO: 16 or SEQ ID NO: 51. In embodiments, the intracellular signaling domain includes the functional signaling domain of 4-1BB and / or the functional signaling domain of CD3 zeta.

[0220] In an embodiment, the intracellular signaling domain includes the sequence of SEQ ID NO: 16 and / or the sequence of SEQ ID NO: 17 or SEQ ID NO: 43. In an embodiment, the CAR further includes a leader sequence. In an embodiment, the leader sequence includes SEQ ID NO: 13.

[0221] In this embodiment, the cells expressing the CAR molecule include T cells or NK cells.

[0222] In embodiments, the disease associated with CD19 expression is selected from proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as spinal cord malformations, myelodysplastic syndromes or preleukemic states, or non-cancer-related signs associated with CD19 expression. In embodiments, the disease is one or more of the following: hematological cancers, acute leukemia, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoblastic leukemia (ALL), chronic leukemia, chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL).

[0223] In embodiments, the method further comprises administering a drug that enhances the efficacy of cells expressing CAR molecules. In embodiments, the method further comprises administering a drug that improves one or more side effects associated with the administration of cells expressing CAR molecules. In embodiments, cells expressing CAR molecules are administered in combination with a drug that treats a CD19-related disease.

[0224] In embodiments of the methods described herein, e.g., methods for providing antitumor immunity to a mammal or methods for treating a mammal, the mammal is a non-responder, partially-responder, or fully-responder to a previously administered cancer treatment, e.g., CD19 CAR therapy or cancer treatment other than CD19 CAR-expressing cells. In embodiments, the mammal is a non-recurrent, partially-recurrent, or fully-recurrent patient to a previously administered cancer treatment, e.g., CD19 CAR therapy or cancer treatment other than CD19 CAR-expressing cells. In embodiments, the mammal has CD19-negative cancer cells or CD19-positive cancer cells, and the mammal may further have CD22-positive, CD123-positive, FLT-3-positive, ROR-1-positive, CD79b-positive, CD179b-positive, CD79a-positive, CD10-positive, CD34-positive, and / or CD20-positive cancer cells. In embodiments, the mammal has recurrent ALL cancer. In embodiments, the mammal has been previously administered CD19 CAR-expressing cells and CD19 CAR treatment is ineffective.

[0225] In embodiments, the drug is an mTOR inhibitor, and the subject is administered a low dose of an immune-enhancing mTOR inhibitor, such as RAD001 or rapamycin. In embodiments, the mTOR inhibitor is RAD001. In embodiments, the dose includes allosteric mTOR inhibitors and catalytic mTOR inhibitors. In embodiments, the mTOR inhibitor is administered for a sufficient time to decrease the ratio of PD-1-positive T cells, increase the ratio of PD-1-negative T cells, or increase the PD-1-negative T cell / PD-1-positive T cell ratio in the peripheral blood of the subject or in a T cell preparation isolated from the subject.

[0226] In embodiments, immune effector cells, e.g., T cells, processed to express CARs were harvested after a sufficient time or after sufficient administration of a low dose of an immune-enhancing mTOR inhibitor, resulting in a temporary increase in the level of PD1-negative immune effector cells, e.g., T cells, or the ratio of PD1-negative immune effector cells, e.g., T cells to PD1-positive immune effector cells, e.g., T cells, in or from a subject. In embodiments, the dose of the mTOR inhibitor was at least 5% but not exceeding 90% mTOR inhibition, as measured by, for example, p70 S6 K inhibition. In embodiments, the dose of the mTOR inhibitor was at least 10% but not exceeding 40% mTOR inhibition, as measured by, for example, p70 S6 K inhibition.

[0227] In one embodiment, the method further includes administering a checkpoint inhibitor. In an embodiment, the subject receives pretreatment with the drug, e.g., an mTOR inhibitor and / or a checkpoint inhibitor, before initiating CART therapy. In an embodiment, the subject receives concurrent treatment with the drug, e.g., an mTOR inhibitor and / or a checkpoint inhibitor. In an embodiment, the subject receives treatment with the drug, e.g., an mTOR inhibitor and / or a checkpoint inhibitor, after CART therapy.

[0228] In the embodiment, the determined level or characteristic is obtained before, simultaneously with, or during CART therapy.

[0229] In embodiments, the method includes assaying a genetic signature indicating whether a subject is likely to relapse or has relapsed. In embodiments, the method includes assaying a genetic signature in a subject that foreshadows relapse to CAR treatment before treatment with CAR-expressing cells, for example, CART treatment (e.g., CART19 treatment, e.g., CTL019 treatment). In embodiments, the level of one or more markers is the level of at least two, three, four, five, six, seven, eight, nine, or ten markers listed in Table 29. In embodiments, the level of a marker includes mRNA levels or soluble protein levels.

[0230] In embodiments, the characteristic of CD19 is a mutation in exon 2, for example, a mutation that causes a frameshift or an immature stop codon or both. In embodiments, T REG At the cellular level, T REG This is determined by staining the sample for markers expressed by cells. In one embodiment, T REG The cellular level refers to the level of Treg cells in the relevant location within the body, for example, in the tumor microenvironment.

[0231] In one embodiment, the method involves T in the object before apheresis. REG The method further includes reducing the signature. In embodiments, the method involves administering, for example, cyclophosphamide, an anti-GITR antibody, or both to the subject, thereby reducing the T in the subject. REG The method further includes reducing the signature. In embodiments, the method includes pre-treating the subject with cyclophosphamide, an anti-GITR antibody, or both, before collecting cells for the production of CAR-expressing cell products. In embodiments, the method further includes obtaining a sample from the subject, the sample including a cell fraction (e.g., including blood), a tissue fraction, an apheresis sample, or a bone marrow sample.

[0232] In the embodiment, the cell expresses an inhibitory molecule comprising a first polypeptide, which comprises at least a portion of the inhibitory molecule, and a second polypeptide, which comprises a positive signal from an intracellular signaling domain. In the embodiment, the inhibitory molecule comprises a first polypeptide comprising at least a portion of PD1 and a second polypeptide comprising a co-stimulatory domain and a primary signaling domain.

[0233] In embodiments, the method includes assaying a genetic signature indicating whether a cell-treated subject is likely to relapse or has relapsed. In embodiments, the method includes assaying a genetic signature in cells before injection into the subject. In embodiments, the method includes T2 of a cell population containing transduced cells. REG Further includes reducing the signature. In an embodiment, T REG Reducing the signature includes implementing CD25 depletion on the cell population.

[0234] In this embodiment, the subject is a mammal, such as a human.

[0235] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Preferred methods and materials are described below, but similar or equivalent methods and materials may be used in the implementation or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein (for example, with respect to sequence database reference numbers) are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referenced herein, for example, in any of the tables herein, are incorporated by reference. Unless otherwise specified, sequence accession numbers specified herein, including those in any tables herein, refer to database entries as of April 8, 2015. If multiple sequence accession numbers are listed for reference for a single gene or protein, all of those sequence variants are included.

[0236] In addition, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0237] Headings, subheadings, or numbered or letter-classified elements, such as (a), (b), (i), etc., are provided solely for readability. The use of headings or numbered or letter-classified elements in this document does not require that the steps or elements be performed in alphabetical order, or that the steps or elements be discontinuous from one another.

[0238] Other features, purposes, and advantages of the present invention are expected to be apparent from the description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0239] [Figure 1] Figures 1 and 1B are schematic diagrams of typical CARs. [Figure 2]Figure 2 includes images of immunohistochemical analysis of Hodgkin lymphoma showing CD19-expressing cells present in the tumor. The left panel is at 1x magnification, and the right panel is at 20x magnification. [Figure 3] Figure 3 is a schematic diagram of the experimental setup for a study to evaluate the therapeutic efficacy of CART19 treatment in patients with Hodgkin lymphoma. [Figure 4] Figures 4A, 4B, 4C, and 4D show flow cytometry analysis of PD1 and CAR19 expression in T cells. Figures 4A and 4B are representative flow cytometry profiles demonstrating the distribution of PD-1 and CAR19 expression in CD4+ T cells from control groups that were either complete response (CR) or non-response (NR) to CART therapy. Figure 4C is a graph showing the percentage of PD1 cells in CD4+ T cell populations from control groups with different responses to CART therapy. Figure 4D is a graph showing the percentage of PD1 cells in CD8+ T cell populations from control groups with different responses to CART therapy. [Figure 5] Figures 5A and 5B show the distribution of PD1 expression in CD4 and CAR19-expressing cells (Figure 5A) or CD8 and CAR19-expressing cells (Figure 5B) from a control group exhibiting different responses to CART therapy. [Figure 6] Figure 6 shows flow cytometry analysis of PD1, CAR19, LAG3, and TIM3 expression in T cells derived from subjects that were either fully responsive (CR) or non-responsive (NR) to CART therapy. [Figure 7] Figures 7A and 7B show the distribution of PD1 and LAG3 expression (Figure 7A) or PD1 and TIM3 expression (Figure 7B) from the control group with different responses to CART therapy. [Figure 8] Figure 8 shows the IgA immunophenotypic analysis of plasma cells from myeloma patients who received CART19, demonstrating the response to CART19 therapy. [Figure 9]Figures 9A and 9B show IL-7 receptor (CD127) expression on cancer cell lines and CART cells. CD127 expression was measured by flow cytometry in three cancer cell lines: RL (mantle cell lymphoma), JEKO (also known as Jeko-1, a mantle cell lymphoma), and Nalm-6 (B-ALL) (Figure 9A). CD127 expression was measured by flow cytometry in circulating CD3-positive (CART) cells injected into NSG mice (Figure 9B). [Figure 10] Figures 10A, 10B, and 10C show the antitumor response after CART19 treatment and subsequent IL-7 treatment. NSG mice engrafted with luciferase-expressing mantle lymphoma cell line (RL-luc) on day 0 were treated with various doses of CART19 cells on day 6, and tumor burden was monitored. Mice were divided into four groups: no CART19 treatment, 0.5 × 10⁶ CART19 cells (CART19 0.5E6), 1 × 10⁶ CART19 cells (CART19 1E6), or 2 × 10⁶ CART19 cells (CART19 2E6). Tumor burden after CART treatment was measured by detection of bioluminescence (mean BLI) (Figure 10A). Mice receiving either 0.5 × 10⁶ CART19 cells (CART19 0.5E6) or 1 × 10⁶ CART19 cells (CART19 1E6) were randomized to receive recombinant human IL-7 (rhIL-7) or not. Here, tumor burden, expressed as mean bioluminescence (BLI), was monitored for three mice from Figure 10A (#3827, #3829, and #3815, who received the indicated initial CART19 dose) treated with IL-7 initiated at day 85 (Figure 10B). IL-7 was administered three times weekly by IP injection. Here, tumor burden, expressed as mean bioluminescence (BLI), at 85 days prior (PRE) and 115 days post (POST) was compared between mice that did not receive IL-7 (CTRL) and mice that received IL-7 (IL-7) (Figure 10C). [Figure 11]Figures 11A and 11B show T cell dynamics after IL-7 treatment. Levels of human T cells detected in the blood were monitored in IL-7-treated mice and control mice, respectively (Figure 11A). Levels of CART19 cells (CD3+ cells) detected in the blood were measured before the start of IL-7 treatment (PRE) and 14 days after (Day 14) (Figure 11B). [Figure 12] Figure 12 illustrates the structures of two example RCAR configurations. The antigen-binding member includes an antigen-binding domain, a transmembrane domain, and a switch domain. The intracellular binding member includes a switch domain, a co-stimulatory signaling domain, and a primary signaling domain. The two configurations demonstrate that the first and second switch domains described herein can be oriented differently with respect to the antigen-binding and intracellular binding members. Other RCAR configurations are further described herein. [Figure 13] Figure 13 shows two constructs for a bispecific CAR having anti-C22 and anti-CD19 binding domains. "4G4S" represents the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 1311). [Figure 14] Figure 14 is a graph showing the activity of the bispecific CD19 / CD22 CAR construct in the NFAT assay. [Figure 15] Figures 15A, 15B, and 15C are graphs showing the degree of CAR T cell activation (measured by relative luminescence) in the presence of various tumor target cell lines. Figure 15A shows CAR T cell activation in the presence of Daudi, a target cell line expressing CD20. Figure 15B shows CAR T cell activation in the presence of Raji, a target cell line expressing CD20. Figure 15C shows CAR T cell activation in the presence of K562, a negative control that does not express CD20. [Figure 16]Figure 16 is an exemplary schematic diagram illustrating the overview of gene signature analysis. Briefly, for each gene set, a two-group statistical model was applied to determine whether the metagenes were statistically different among CR, PR, and NR. CR closely resembles resting TEFF cells, while NR closely resembles activated TEFF cells. Genes upregulated in activated and resting TEFF cells are also upregulated in NR. [Figure 17] This study exemplifies the high expression level of the TREG gene in samples from pediatric patients who were complete responders but experienced relapse (R), compared to complete responders (CR) who did not experience relapse (p=0.000215). The x-axis represents samples from the response group, where CR = complete responders without relapse and R = responders. The y-axis represents the standardized metagene expression score. [Figure 18] Figures 18A, 18B, and 18C are graphs showing CAR T cell activation in the presence of tumor target cell lines. In Figure 18A, CAR-expressing JNL cells were mixed with a target cell line expressing Daudi CD22 at the indicated E-to-T ratio. In Figure 18B, CAR-expressing JNL cells were mixed with a target cell line expressing Raji CD22 at the indicated E-to-T ratio. In Figure 18C, CAR-expressing JNL cells were mixed with the negative control K562 cell line at the indicated E-to-T ratio. [Figure 19-1] Figure 19 is a graph showing the primary T cell expression of chimeric antigen receptors on the cell surface. CAR surface expression levels were determined using protein-L-biotin / SA-PE (Figures 19-1 and 19-2) and rhCD22-Fc / anti-Fc488 (Figures 19-3 and 19-4). Cells lacking CAR were used as negative controls. [Figure 19-2] Same as above [Figure 19-3] Same as above [Figure 19-4] Same as above [Figure 20]Figures 20A, 20B, 20C, 20D, 20E, and 20F are graphs showing primary T cell tumor target death assays. Primary T cells activated and transduced with CD22 CARs were mixed with target cell lines stably expressing luciferase at the indicated ratios, and target cell death was measured. Death rates were standardized against hCD22-8 (28.8% transduced). Functional CD22 CAR clones were tested against positive control CD22 CAR m971 (m971-HL), negative control CAR m971-LH, and non-transduced T cells as negative control, using CD22-expressing cell lines Raji (Figure 20A), SEM (Figure 20B), K562-hCD22 (Figure 20C), Daudi (Figure 20D), and Nalm6 (Figure 20E). The K562 cell line did not express CD22 and was used as a negative control (Figure 20F). [Figure 21-1] Figures 21A, 21B, 21C, 21D, 21E, and 21F are graphs showing the induction of a significant pro-inflammatory cytokine response by CD22 CAR clones. The ability of CD22 CAR clones to produce the pro-inflammatory cytokines IFN-γ, IL-2, and TNFα was determined using a primary T cell death assay. Effector cells were co-cultured with each of the different target cell lines for 20 hours and normalized to 28.8% transduction. The supernatant was collected from different cultures of target cells expressing Raji CD22 (Figure 21A), Nalm6 CD22 (Figure 21B), Daudi CD22 (Figure 21C), SEM CD22 (Figure 21D), K562-hCD22 (Figure 21E), and K562 non-CD22 expressing cells (negative control) (Figure 21F), with varying E-to-T ratios of 2.5:1 and 10:1. [Figure 21-2] Same as above [Figure 21-3] Same as above [Figure 22]Figure 22 is a graph showing the expression of various B cell antigens in relapsed ALL as detected by flow cytometry. Samples from 16r / r patients were screened by multiparametric flow cytometry for the following markers: CD19 (16pts), CD22 (16pts), CD123 (16pts), FLT-3 (9pts), ROR-1 (3pts), CD79b (15pts), CD179b (8pts), CD79a (16pts), CD10 (16pts), CD34 (16pts), and CD20 (16pts). CD22 and CD123 were highly (>60%) and homogeneously expressed in blast cells from r / r ALL patients (bars indicate median expression percentages, 99.50%, 98.80%, 95.70%, 72.00%, 47.00%, 15.00%, 13.45%, 4.200%, 98.00%, 87.65%, and 7.00%, respectively). For each patient, the percentage of cells expressing the indicated markers is shown as a single data point. [Figure 23] Figure 23 shows a series of graphs illustrating the expression of CD22 and CD123 in six patients with relapsed CD19-negative leukemia, both before (baseline) and after (CD19-negative relapse) CART19 treatment. In all analyses, the target population was gated based on forward-to-side scattering characteristics followed by singlet gating, and viable cells were gated using Live Dead Aqua (Invitrogen). Time gating was included for quality control. The gating strategy included time gating → low SSC → singlet → viable → CD45 dim → CD10+. [Figure 24] Figure 24 is a series of graphs showing CD22 expression in blast cells from patients with relapsed CD19-negative disease after CART19 treatment (clinical trial UPCC04409 / CHP959, patient UPNs are shown in square boxes). The top column shows CD19 and CD22 expression in blast cells before CART19 treatment, while the bottom column shows the disease phenotype at relapse. CD22 expression was also maintained at relapse when CD19 expression was lost. [Figure 25]Figure 25 is a set of graphs showing CD123 expression in blast cells from patients with relapsed CD19-negative disease after CART19 treatment (clinical trial UPCC04409 / CHP959, patient UPNs are shown in square boxes). The top column shows CD19 and CD123 expression in blast cells before CART19 treatment, while the bottom column shows the disease phenotype at relapse. CD123 expression was maintained at relapse in most patients, while CD19 expression was lost. [Figure 26] Figure 26 is a graph showing the median expression levels of CD19, CD22, and CD123 before and after CART19 treatment in patients with relapsed CD19-negative disease. CD19 expression was lost at relapse (94.25% vs. 0%, p=0.0009), while CD22 (99.20% vs. 97.30%, p=ns) and CD123 (63.00% vs. 48.75%, p=ns) remained expressed. For each patient, the percentage of cells expressing the indicated marker is shown as a single data point. [Figure 27] Figures 27A and 27B are a series of graphs showing CD22 expression in samples from 16 r / r patients and samples from 4 patients who relapsed with CD19-negative disease after CART19 treatment. Samples were screened for the B-cell marker CD22 by multiparametric flow cytometry. CD22 was highly (>60%) and homogeneously expressed in blast cells of 11 out of 15 r / r ALL patients (Figure 27A). CD22 was positive both before (baseline) and after (CD19-negative relapse) CART19 treatment (showing 2 points) in all 4 patients who relapsed with CD19-negative leukemia (Figure 27B). Gating strategy: low SSC → singlet → survival → CD45 dim. [Figure 28]Figures 28A, 28B, and 28C are a series of graphs showing the effect of CD22 CART on CD19 and CD22 expression. An overview of two CAR22 constructs generated using different chain orientations (H to L and L to H) is shown (Figure 28A). Anti-CD22 scFv(m971) was codon-optimized and cloned into a mouse CAR19 vector containing the CD8 hinge, 41-BB costimulation, and CD3 zeta signaling domain (Figure 28A). CD19, CD22, and isotype control expression in the NALM6 ALL cell line are shown as mean fluorescence intensity (MFI) (Figure 28B) and antibody binding capacity (ABC) (Figure 28C). In NALM-6, CD19 expression was higher than CD22. However, in most primary ALL samples, CD19 and CD22 expression were similar (see Figure 27A). [Figure 29] Figures 29A, 29B, and 29C are a series of graphs showing the proliferation of normal donor T cells to generate CART22 and CART19 (along with UTD cells). Population doubling (PD) vs. days of culture: At the end of proliferation (day 11), CART22 and control T cells reached approximately 4.5 PD, with no significant difference compared to CART19 or UTD cells (Figure 29A). T cell volume (fl) vs. days of culture: Peak volume (approximately 450 fl) was reached on day 6, while the volume decreased to 300 fl the following day when the cells were harvested and frozen. No significant difference was observed compared to CART19 or UTD cells (Figure 29B). CAR expression of CD4-positive and CD8-positive T cells on day 11 of proliferation is shown in Figure 29C. Gating for CAR expression is based on UTD. Gating strategy: FSS vs. SSC lymphocytes → singlet → survival → CD3+. [Figure 30]Figure 30 shows a series of graphs illustrating the CD107a degranulation assay with intracellular cytokine production. CART19, CART22 HtoL, and LtoH were co-cultured with different targets (alone, PMA / ionomycin, MOLM-14, and NALM-6). CART19 and CART22 HtoL showed high levels of CD107a degranulation, IL-2, IFNg, and TNFa production when co-cultured with the ALL cell line (NALM-6) and not with a negative control. UTD and CART22 LtoH did not show degranulation or cytokine production. Gating strategy: FSS vs. SSC lymphocytes → singlet → survival → CD3+. [Figure 31] Figure 31 is a graph showing the luciferase-based cell death assay. CART22 and CART19 HtoL, rather than UTD cells, were able to lyse NALM-6 cells after 24 hours of co-culture. A direct correlation was observed between cytotoxic activity and the E-to-T ratio, with a 2:1 E-to-T ratio (78% and 75% cell death for CART19 and CART22) indicating better antileukemic efficacy. [Figure 32] Figures 32A and 32B are a series of graphs showing CFSE-based proliferation assays. Five-day co-culture of CART22 and CART19 cells using the ALL cell line NALM-6 resulted in significant T cell proliferation (94% and 92.9%, respectively). Controls are also shown (TCM = medium only, PI = PMA / ionomycin, MOLM-14) (Figure 32A). Histograms showing the dynamics of CFSE dilution in CART19 and CART22 show that most T cells underwent multiple proliferation cycles (Figure 32B). Gating strategy: FSS vs. SSC lymphocytes → singlet → survival → CD3+. [Figure 33]Figure 33 shows a series of graphs illustrating cytokine production. CART22, CART19, and UTD were incubated for 24 hours with different irradiation targets (alone, PMA / ionomycin, MOLM-14, and NALM-6). When co-cultured with the ALL cell line NALM-6, only CART22 and CART19HtoL were able to release multiple cytokines (shown here as IFNg, IL-2, GM-CSF, TNFα, and MIP1b). Results are shown as mean intensity fluorescence (MFI). [Figure 34] Figures 34A and 34B are a series of graphs showing T cell degranulation with primary ALL blast cells. CART22, CART19, and UTD cells were co-incubated for 4 hours with ALL patient-derived blast cells (CHP-959-101) at baseline and after CART19 treatment when patients relapsed with CD19-negative disease. Both CART19 and CART22 were able to degranulate at baseline (when blast cells were CD19+ and CD22+), but only CART22 degranulated at relapse (when the disease was CD19-negative) (Figure 34A). Dot plots showing CD107a degranulation in CD8-positive and CD8-negative CART19 and CART22 effectors after incubation with CHP101 samples at relapse demonstrated only CART22 showing degranulation in both CD8 and CD4 T cells (Figure 34B). Gating strategy: FSS vs. SSC lymphocytes → singlet → survival → CD3+. [Figure 35-1]Figures 35A, 35B, 35C, and 35D are a series of graphs showing the in vivo efficacy of CART22 against NALM-6. A. Experimental Summary: 1 million NALM-6 luciferase+ cells / mouse were IV injected into NSG mice. After 6 days, tumor engraftment was assessed by bioluminescence. Next, mice were randomized to receive either non-transduced T cells or different doses of CART22 (1.25 to 5 million total cells / mouse, 75% CAR expression). Mice were then monitored for tumor load, PB T cell proliferation, and survival (Figure 35A). Tumor load by bioluminescence (BLI) detected a dose-related anti-leukemic response. Mice receiving 5e6 CART22 cells showed better tumor control (Figure 35B). Mice treated with CART22 showed statistically significantly better overall survival (OS) compared to mice treated with UTD cells. Regarding overall survival (OS), there was a significant correlation between higher doses of CART22 and better OS (Figure 35C). In vivo T cell proliferation was monitored weekly by retroorbital hemorrhage. One week after T cell infusion, mice receiving high doses of CART22 showed better CART expansion (median 12 T cells / μl) (Figure 35D). [Figure 35-2] Same as above [Figure 36-1]Figures 36A and 36B are a series of graphs showing an in vivo comparison between CART22 and CART19 for NALM-6. Experimental summary: 1 million NALM-6 luciferase+ cells / mouse were IV injected into NSG mice. After 6 days, tumor engraftment was assessed by bioluminescence. Mice were then randomized to receive non-transduced T cells, CART19, or CART22 (5 million total cells, 75% CAR expression). Mice were then monitored for tumor load, PB T cell proliferation, and survival (Figure 36A). Tumor load by bioluminescence (BLI) showed an anti-leukemic response in mice treated with both CART22 and CART19, while UTD mice progressed rapidly (Figure 36B). Presumably due to different target expression in NALM-6 (CD19>>CD22), CART19-treated mice showed better overall survival (OS) compared to CART22 mice (Figure 36C). [Figure 36-2] Same as above [Figure 37-1] Figures 37A and 37B are a series of graphs showing an in vivo comparison between CART22 and CART19 in a model of primary ALL. Blast cells (JH331) from first-stage ALL patients were passaged in vivo and transduced with luciferase to track tumor burden. Experimental summary: 1 million JH331 luciferase+ cells / mouse were IV injected into NSG mice. After 14 days, tumor engraftment was assessed by bioluminescence. Mice were then randomized to receive untransduced T cells, CART19, or CART22 (5 million total cells, 75% CAR expression). Mice were monitored for tumor burden, PB T cell proliferation, and survival (Figure 37A). Tumor burden by bioluminescence (BLI) detected an anti-leukemic response in mice treated with both CART22 and CART19, while UTD mice progressed rapidly (Figure 37B). [Figure 37-2] Same as above [Figure 38-1]Figures 38A, 38B, and 38C are a series of images showing tissue microarrays of CD22 expression in 28 normal human tissues, as determined by immunohistochemical staining. Lymphoid organs were positive for CD22 expression (tonsils, lymph nodes, spleen, and thymus) (Figure 38A). Non-lymphoid organs did not show CD22 expression (Figure 38B). CD22-positive resident B cells were observed in multiple tissues (Figure 38C). * = Nonspecific staining. [Figure 38-2] Same as above [Figure 38-3] Same as above [Figure 39-1] Figure 39 is a graph showing CD22 RNA expression data from GeneAtlas U133A. High levels of CD22 expression were observed in B cells, tonsils, and lymph nodes. B lymphoblasts and leukemia / lymphoma cell lines were also highly positive. [Figure 39-2] Same as above [Figure 39-3] Same as above [Figure 39-4] Same as above [Figure 40] Figure 40 shows a series of graphs illustrating the 51-chromium release assay for CART22 toxicity. Both CART22 and CART19 induced lysis of the ALL cell line NALM-6, but not in UTD cells. No cytotoxic effects of CART22 were observed in either normal tissue (CD34+, human neuron precursors or neurons and keratinocytes) or control (K562 cell line). [Figure 41] Figure 41 shows a graphical representation of CAR expression in JNL cells transduced with the anti-CD123 CAR construct, as evaluated by FACS and reported as the percentage of cells showing a signal above the signal level in non-transduced (CAR-negative) cells using protein L as the detection reagent. [Figure 42]Figures 42A, 42B, and 42C show graphs of CD123 CAR activity in JNL cells. The activity of anti-CD123 CAR constructs was evaluated using Jurkat cell lines (referred to as JNL cells) containing a luciferase reporter driven by the NFAT promoter. CAR activity is measured as activation of this NFAT-driven reporter. [Figure 43] Figures 43A and 43B show CD123 expression and activity. Figure 43A shows a graphical representation of CD123 CAR expression in primary T cells. The percentage of transduced cells (expressing anti-CD123 CAR on the cell surface) and the relative fluorescence intensity of their expression were determined by flow cytometry analysis in BD LSRFortessa or BD-FACSCanto using protein L as the detection reagent. A gating histogram plot of the relative fluorescence intensity from its FACS relative to the signal on unstained cells shows the percentage of transduced T cells. Transduction resulted in a range of 12–42% CAR-positive cells. Figure 43B shows a graphical representation of CD123-CAR-mediated cell death. T cell death was directed at CD123-expressing MOLM13 acute myeloid leukemia cells that stably express luciferase. Non-transduced T cells were used to determine nonspecific background death levels. The cytolytic activity of CART-CD123 was measured with an effector-to-target cell ratio of 4:1 and over a range of 2-fold downward dilution of T cells, in which case the effector was defined as T cells expressing the anti-CD123 chimeric receptor. The assay was initiated by mixing an appropriate number of T cells with a fixed number of target cells. After 20 hours, luciferase signaling was measured using the Bright-Glo® luciferase assay on an EnVision instrument. [Figure 44] Figures 44A and 44B show the transduction efficiency of T cells using CD123-CARs. Figure 44A shows the transduction efficiency of T cells using 1172 and 1176. Figure 44B shows the transduction efficiency of T cells using CD123 CARs 2-4. [Figure 45]Figure 45 shows flow cytometry of CD123 CARs 2-4 and 1172 and 1176 to determine the CD4 to CD8 ratio. [Figure 46] Figure 46 shows the degranulation of CD123 CARs 2-4 and 1172 and 1176 upon exposure to CD123+ tumor cells. [Figure 47] Figure 47 shows a graphical representation of a luciferase assay to evaluate the cytotoxicity of CART cells (NVS 2-4, 1172 and 1176 clones) against tumor target cells (MOLM14). [Figure 48] Figure 48 shows a comparison of tumor burden in NSG mice injected with luciferase-expressing MOLM14 cells on day 6 (before CART injection) and day 13 (6 days after injection with NVS 2-4, 1172, or 1176 clones) or day 20. [Figure 49-1] Figures 49A, 49B, 49C, 49D, 49E, and 49F show that CD123 is highly expressed in relapses of CD19-negative B-cell acute lymphoblastic leukemia occurring after CART19 treatment. Figure 49A shows the expression of CD123 compared to CD19 in 42 relapsed / refractory ALL samples. Figure 49B shows the co-expression of CD123 and CD19 in B-ALL blasts. Blasts are gated (low SSC, singlet, viable, CD45dim). Figure 49C shows the gating strategy of leukemia stem cells (LSCs). CD123 is highly expressed in this subset. Figure 49D shows the co-expression of CD123 and CD19 and the results of FISH analysis. Figures 49E and 49F show a comparison of CD19 and CD123 expression at baseline or after relapse. [Figure 49-2] Same as above [Figure 49-3] Same as above [Figure 50-1]Figures 50A, 50B, 50C, 50D, 50E, and 50F show the results of various in vitro assays using T cells expressing CD19 CAR (CAR19) or CD123 CAR (CAR123). Figure 50A shows the expression of CD19 and CD123; Figure 50B shows the CD107a degranulation assay; Figure 50C shows the ability to target cell death; Figures 50D and 50E show the proliferative capacity; and Figure 50F shows cytokine production for the indicated cytokines. [Figure 50-2] Same as above [Figure 51] Figures 51A, 51B, and 51C demonstrate that CART cells expressing CD19 CAR (CAR19) or CD123 CAR (CAR123) exhibit antitumor effects in an in vivo mouse model. Figure 51A shows tumor burden as represented by bioluminescence imaging; Figure 51B shows the overall survival curve of mice treated with CART therapy; and Figure 51C shows proliferation of CART123 cells in peripheral blood. [Figure 52-1] Figures 52A, 52B, 52C, 52D, 52E, and 52F demonstrate that CART123 is active in an in vivo mouse model of antigen-deficient relapse. Figure 52A shows an experimental overview; Figure 52B shows disease progression represented by bioluminescence imaging in baseline and relapsed disease (upper graph) and in response to treatment with CART19 therapy (lower graph) for CD19 expression; Figure 52C shows bioluminescence images of mice administered with non-transduced T cells or CART19 cells; Figure 52D shows an experimental overview for treatment with CART19 or CART123; Figure 52E shows disease progression; Figure 52F shows the overall survival rate of treated mice. [Figure 52-2] Same as above [Figure 52-3] Same as above [Figure 52-4] Same as above [Figure 53-1]Figures 53A, 53B, and 53C show ALL-CART interactions in the skull bone marrow of xenograft mice. Figure 53A shows an experimental overview; Figure 53B shows representative multiphoton XY planar images of CART19 and CART123 cells interacting with ALL tumors, genetically engineered to express either CD19 and CD123 or CD123 alone (motile cells are indicated by dashed circles, and non-motile cells by arrows); Figure 53C is a graphical representation of the microscopic images. [Figure 53-2] Same as above [Figure 54-1] Figures 54A, 54B, and 54C demonstrate the prevention of CD19-negative relapse using CART19 and CART123. Figure 54A shows an overview of the experiment; Figure 54B shows disease progression (tumor burden, represented by BLI) in mice treated with non-transduced T cells (top graph), CART19 (middle graph), or a combination of CART19 and CART123 (bottom graph); Figure 54C shows the overall survival rate from this experiment. [Figure 54-2] Same as above [Figure 55] Figures 55A and 55B show T cells expressing both CAR19 and CAR123 (Figure 55A) and the results of a degranulation assay (Figure 55B). [Figure 56] Figures 56A and 56B show the characterization of ALL blast cells. Figure 56A shows the expression of various markers, CD19, CD123, CD10, CD34, and CD20; Figure 56B shows a gating strategy for sorting CD19-CD123+ cells. [Figure 57] Figures 57A, 57B, 57C, and 57D show the anti-leukemic activity of CART123. Figure 57A shows the expression of CD19 and CD123 in NALM6 cells; Figure 57B shows tumor burden (represented by BLI) in response to CART19 or CART123 therapy; Figure 57C shows the overall survival of mice administered with CART19 or CART123; Figure 57D shows the overall survival of mice administered with various doses of CART123. [Figure 58]Figures 58A and 58B illustrate the characterization of an in vivo model of antigen-loss relapse. Figure 58A shows CD123 expression in CD19-negative relapsed disease; Figure 58B shows a degranulation assay of CART19 or CART123 cells in vitro when cultured with baseline cells or relapsed cells. [Figure 59] Figure 59 shows that the proliferation of CAR-expressing transduced T cells is enhanced by low doses of RAD001 in a cell culture system. CART cells were co-cultured with NALM6 (Nalm-6) cells in the presence of different concentrations of RAD001 (nM). The number of CAR-positive CD3-positive T cells (black) and total T cells (gray) was assessed 4 days after co-culture. [Figure 60] Figure 60 illustrates tumor growth measurements of NALM6-luc cells with daily administration or vehicle administration of RAD001 at 0.3, 1, 3, and 10 mg / kg (mpk). Circles represent vehicle administration, squares represent 10 mg / kg dose of RAD001, triangles represent 3 mg / kg dose of RAD001, inverted triangles represent 1 mg / kg dose of RAD001, and diamond shapes represent 0.3 mg / kg dose of RAD001. [Figure 61] Figures 61A and 61B show pharmacokinetic curves illustrating the amount of RAD001 in the blood of NSG mice with NALM6 tumors. Figure 61A shows the PK on day 0 following the first dose of RAD001. Figure 61B shows the PK on day 14 following the final dose of RAD001. Diamonds represent a 10 mg / kg dose of RAD001, squares represent a 1 mg / kg dose of RAD001, triangles represent a 3 mg / kg dose of RAD001, and x represents a 10 mg / kg dose of RAD001. [Figure 62]Figures 62A and 62B show in vivo proliferation of humanized CD19 CART cells with and without RAD001 administration. Low daily doses of RAD001 (0.003 mg / kg) enhance CAR T cell proliferation beyond normal levels of huCAR19 proliferation. Figure 62A shows CD4+ CAR T cells; Figure 62B shows CD8+ CAR T cells. Circles represent PBS, squares represent huCTL019, triangles represent huCTL019 with 3 mg / kg RAD001, inverted triangles represent huCTL019 with 0.3 mg / kg RAD001, diamonds represent huCTL019 with 0.03 mg / kg RAD001, and black circles represent huCTL019 with 0.003 mg / kg RAD001. [Figure 63] Figure 63 shows multiple FIHC AQUA analyses demonstrating significant differences between CD3+ / PD-1+ cell populations in primary and secondary human DLBCL patient samples. [Figure 64] Figure 64 shows AQUA analysis of various levels of CD19 (lower panel) and PD-L1 (upper panel) in primary and secondary sites of DLBCL samples. A total of 40 human DLBCL patient samples, 25 primary sites and 15 secondary sites, were subjected to multiplex FIHC, followed by AQUA analysis to identify the expression levels of CD19 and PD-L1 proteins. [Figure 65] Figure 65 shows schematic diagrams of two populations of CAR-expressing cells. In the population on the left (pooled), each cell expresses one type of CAR. In the population on the right (two-cistronic CARs), each cell expresses two types of CARs. [Figure 66] Figure 66 shows a diagram of a bicistronic CAR. The upper CAR has CD19 CAR and CD22 CAR separated by the P2A protease cleavage site. The lower CAR has CD19 CAR and CD123 CAR separated by the P2A protease cleavage site. [Figure 67] Figure 67 shows the simultaneous expression of CD19 and CD22 CARs from a bisistronic vector. [Figure 68]Figure 68 shows the co-expression of CD19 and CD123 CARs from a bicistronic vector. The lower panel of Figure 68 shows the anti-leukemic effects of these cells. [Figure 69] Figure 69 shows the tumor burden in mice with CD19-negative B-ALL xenografts after treatment with UTD control, CART19, or CART22. [Figure 70] Figure 70 shows the expression of PD-L1, PD1, LAG3, and TIM3 (from left to right in each set of four bars) in lymph node and bone marrow samples from five CR patients, one unclassified patient, and six PD patients. [Figure 71] Figure 71 is a graph showing the activation (RLU) of several CD22 CAR constructs in the presence and absence of the m971 competitor. [Figure 72] Figure 72 is a graph showing the activation (RLU) of additional CD22 CAR constructs. [Figure 73] Figure 73 shows three bar graphs illustrating CD22 CAR activity in an IFN-γ assay. [Figure 74] Figure 74 shows the binding activity of CD22-64 and CD22-65 CARs. [Figure 75] Figure 75 is a diagram mapping epitopes coupled by various CD22 scFvs. [Modes for carrying out the invention]

[0240] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention relates.

[0241] The terms "a" and "an" refer to the grammatical object being one or more (i.e., at least one). For example, "an element" means one or more elements.

[0242] The term "approximately" when referring to measurable values ​​such as quantities, temporary durations, and similar items means to include a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1%, from the specified value, because such variation is appropriate for performing the disclosed method.

[0243] The term "apheresis," as used herein, refers to an extracorporeal process recognized in the art of removing donor or patient blood from the donor or patient, passing it through a device that separates and removes specific components of selection, and returning the remainder to the donor or patient's circulation, for example, by reinfusion. Accordingly, "apheresis sample" refers to a sample obtained using apheresis.

[0244] The term “biologically equivalent” refers to the amount of a drug other than the reference compound (e.g., RAD001) required to produce an effect equivalent to the effect produced by the reference dose or amount of the reference compound (e.g., RAD001). In one embodiment, the effect is the level of mTOR inhibition, for example, when measured by P70S6 kinase inhibition, when evaluated by an assay, for example, an assay described herein, such as the Boulay assay, or when measured by measuring the level of phosphorylated S6 by Western blotting. In one embodiment, the effect is a change in the ratio of PD-1 positive / PD-1 negative T cells, when measured by cell sorting. In one embodiment, a bioequivalent amount or dose of the mTOR inhibitor is the amount or dose that achieves the same level of P70S6 kinase inhibition achieved by the reference dose or amount of the reference compound. In one embodiment, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of change in the PD-1-positive / PD-1-negative T cell ratio as the reference dose or reference amount of the reference compound achieves.

[0245] The term “inhibition” or “inhibitor” encompasses a reduction in a particular parameter, e.g., a reduction in the activity of a given molecule, e.g., CD20, CD10, CD19, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. For example, inhibition of activity, e.g., inhibition of at least 5%, 10%, 20%, 30%, or 40% of the activity of CD20, CD10, CD19, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, is included by this term. Therefore, inhibition does not need to be 100%. The activity of an inhibitor may be determined as described herein or by assays known in the art. A "B cell inhibitor" is a cell that causes a decrease in a specific parameter of a B cell, such as activity, such as growth or proliferation, or a specific parameter of a B cell-related molecule, such as a small molecule, antibody, CAR, or a cell containing a CAR. Non-limiting examples of B cell-related molecules include proteins expressed on the surface of B cells, such as CD20, CD10, CD19, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0246] The term “chimeric antigen receptor” or, instead, “CAR” refers to a set of polypeptides, typically two sets of polypeptides in the simplest embodiment, that, when present in an immune effector cell, impart specificity to a target cell, typically cancer cells, and the generation of intracellular signals to that cell. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as the “intracellular signaling domain”) containing a functional signaling domain derived from a stimulating and / or co-stimulating molecule, as defined below. In some embodiments, the sets of polypeptides are on the same polypeptide chain, e.g., a chimeric fusion protein. In some embodiments, the sets of polypeptides are not contiguous with each other, e.g., on different polypeptide chains. In some embodiments, the sets of polypeptides include a dimerization switch that can couple polypeptides with each other in the presence of a dimerizing molecule, e.g., the antigen-binding domain can be coupled to the intracellular signaling domain. In one embodiment, the stimulating molecule of the CAR is a zeta chain (e.g., CD3 zeta) associated with a T cell receptor complex. In one embodiment, the cytoplasmic signaling domain includes a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one embodiment, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one embodiment, the co-stimulatory molecule is selected from the co-stimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27 and / or CD28. In one embodiment, the CAR includes a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR includes 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 embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding 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 stimulator. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding 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 stimulator. In one embodiment, the CAR comprises an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence may be cleaved from the antigen-binding domain (e.g., scFv) during intracellular processing and localization of the CAR to the cell membrane.

[0247] When used herein, the phrase “diseases associated with CD20 expression” includes, for example, proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as spinal malformations, myelodysplastic syndromes, or preleukemic states, as well as conditions related to cells that express CD20 (e.g., wild-type or mutant CD20) or that have expressed CD20 (e.g., wild-type or mutant CD20) at any given time; or non-cancerous signs related to cells that express CD20 (e.g., wild-type or mutant CD20). To avoid misunderstanding, diseases associated with CD20 expression may include, for example, conditions related to cells that previously expressed CD20 but do not currently express CD20 because CD20 expression is downregulated, for example, due to treatment with a CD20-targeting molecule, such as CD20CAR. In one embodiment, cancers associated with CD20 expression are hematological cancers. In one embodiment, hematological cancers include, but are not limited to, AML, myelodysplastic syndrome, ALL, hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia, Hodgkin lymphoma, neoplasms and allogenes of blastic plasmacytoid dendritic cells. Furthermore, diseases associated with CD20 expression include, but are not limited to, atypical and / or non-typical cancers, malignancies, precancerous conditions or proliferative disorders associated with CD20 expression. Non-cancer-related signs associated with CD20 expression may also be mentioned. In some embodiments, CD20-expressing cells express CD20 mRNA or have expressed it at some point in time. In some embodiments, CD20-expressing cells produce CD20 protein (e.g., wild-type or mutant), and this CD20 protein may be present at normal levels or at reduced levels. In some embodiments, CD20-expressing cells produced detectable levels of CD20 protein at some point in time, but subsequently did not produce substantially detectable levels of CD20 protein.

[0248] When used herein, the phrase “diseases associated with CD22 expression” includes, for example, proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as spinal malformations, myelodysplastic syndromes, or preleukemic states, as well as conditions related to cells that express CD22 (e.g., wild-type or mutant CD22) or that have expressed CD22 (e.g., wild-type or mutant CD22) at any given time; or non-cancerous signs related to cells that express CD22 (e.g., wild-type or mutant CD22). To avoid misunderstanding, diseases associated with CD22 expression may include, for example, conditions related to cells that previously expressed CD22 but do not currently express CD22 because CD22 expression is downregulated, for example, due to treatment with a CD22-targeting molecule, such as CD22 CAR. In one embodiment, cancers associated with CD22 expression are hematological cancers. In one embodiment, hematological cancers include, but are not limited to, AML, myelodysplastic syndrome, ALL, hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia, Hodgkin lymphoma, neoplasms and allogenes of blastic plasmacytoid dendritic cells. Furthermore, diseases associated with CD22 expression include, but are not limited to, atypical and / or non-typical cancers, malignancies, precancerous conditions or proliferative disorders associated with CD22 expression. Non-cancer-related signs associated with CD22 expression may also be mentioned. In some embodiments, CD22-expressing cells express CD22 mRNA or have expressed it at some point in time. In some embodiments, CD22-expressing cells produce CD22 protein (e.g., wild-type or mutant), and this CD22 protein may be present at normal levels or at reduced levels. In some embodiments, CD22-expressing cells produced detectable levels of CD22 protein at some point in time, but subsequently did not produce substantially detectable levels of CD22 protein.

[0249] As used herein, unless otherwise specified, the terms “prevent,” “prevent,” and “prevent” refer to actions taken before the subject begins to suffer from the condition or before a recurrence of the condition. Prevention does not have to result in complete prevention of the condition; partial prevention or mitigation of the condition or its symptoms, or a reduction in the risk of developing the condition, are included in this term.

[0250] "Combined" administration, as used herein, means that two (or more) different treatments are delivered to a subject while the subject is suffering from a disorder, for example, two or more treatments are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated or the treatment is discontinued for any other reason. In some embodiments, the delivery of one treatment is still taking place when the delivery of a second treatment begins, so as to be redundant with respect to the administration. This may also be referred to herein as "simultaneous" or "parallel delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of any case, the treatments are more effective by combining the administrations. For example, the second treatment is more effective if it reduces symptoms to the same extent as if the second treatment had not been given, or to the extent that it would be expected to be seen if the second treatment had been administered in the absence of the first treatment, or to the extent that a similar situation would be seen using the first treatment. In some embodiments, the delivery is such that the reduction in other parameters relating to symptoms or impairment is greater than the reduction in parameters expected to be observed with one treatment delivered in the absence of the other. The effects of the two treatments may be partially additive, entirely additive, or more than additive. The delivery may be such that the effect of the first treatment delivered is still detectable when the second is delivered. In one embodiment, CAR-expressing cells are administered in the doses and / or administration schedule described herein, and B-cell inhibitors or agents that enhance the activity of CD19 CAR-expressing cells are administered in the doses and / or administration schedule described herein.

[0251] The term "derived from" indicates the relationship between a first molecule and a second molecule when used herein. This generally refers to the structural similarity between the first and second molecules and does not imply or include any limitation or inclusion of a process or source in the first molecule from which the second molecule is derived. For example, in the case of an intracellular signaling domain derived from the CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure to have the required function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include any limitation to a specific process that produces the intracellular signaling domain, and does not mean, for example, that an intracellular signaling domain must be obtained by starting with a CD3 zeta sequence and deleting or mutating unwanted sequences in order to provide an intracellular signaling domain.

[0252] The term "signaling domain" refers to the functional portion of a protein that acts by transmitting information within an cell to regulate cellular activity via a defined signaling pathway, either by generating a second messenger or by functioning as an effector in response to such a messenger.

[0253] As used herein, the term “CD19” refers to the surface antigen classification 19 protein, which is an antigenic determinant detectable on leukemia progenitor cells. Human and mouse 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 under UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098. As used herein, “CD19” encompasses the protein including mutations in full-length wild-type CD19, such as point mutations, fragments, insertions, deletions, and splice variants. CD19 is expressed in most lineage B cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Other cells that express CD19 are provided in the definition of "diseases associated with CD19 expression" below. This is also an early marker for B cell precursors. See, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one embodiment, the antigen-binding portion of CART recognizes and binds to an antigen within the extracellular domain of the CD19 protein. In one embodiment, the CD19 protein is expressed on cancer cells.

[0254] As used herein, the term “antibody” refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multiple or single-chain, or intact immunoglobulins, and may be of natural or recombinant origin. Antibodies may also be tetramers of immunoglobulin molecules.

[0255] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an antigen's epitope (e.g., by binding, steric hindrance, stabilization / destabilization, or spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv(sdFv); Fd fragments consisting of VH and CH1 domains; linear antibodies; single-domain antibodies, e.g., sdAb(either VL or VH); camelized VHH domains; multispecific antibodies formed from antibody fragments such as bivalent fragments containing two Fab fragments linked by disulfide crosslinking at a hinge region; and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments may also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0256] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are sequentially linked via, for example, a synthetic linker, for example, a short, flexible polypeptide linker, and can be expressed as a single-chain polypeptide, and furthermore, the scFv retains the specificity of the intact antibody from which it originated. Unless otherwise specified, as used herein, the scFv may have the VL and VH variable regions in either order, and, for example with respect to the N-terminus and C-terminus of the polypeptide, the scFv may contain a VL-linker-VH or a VH-linker-VL.

[0257] The term “complementarity-determining region” or “CDR,” as used herein, refers to the amino acid sequence within the antibody variable region that confers antigen specificity and binding affinity. For example, typically there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using one of numerous well-known schemes, such as those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), or combinations thereof. According to the Kabat numbering scheme, in some embodiments, the amino acid residues of the CDR in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the amino acid residues of the CDR in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to the Chothia numbering scheme, in some embodiments, the amino acids of the CDR in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues of the CDR in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In combinations of the Kabat and Chothia numbering schemes, in some embodiments, a CDR corresponds to an amino acid residue that is part of the Kabat CDR, the Chothia CDR, or both.For example, in some embodiments, CDR corresponds to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in VH, e.g., mammalian VH, e.g., human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in VL, e.g., mammalian VL, e.g., human VL.

[0258] As used herein, the terms “binding domain” or “antibody molecule” refer to a protein, such as an immunoglobulin chain or a fragment thereof, that contains at least one immunoglobulin variable domain sequence. The terms “binding domain” or “antibody molecule” encompass antibodies and antibody fragments. In one embodiment, the antibody molecule is a multispecific antibody molecule, for example, comprising a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity to a first epitope, and the second immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity to a second epitope. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity to two or fewer antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules of their naturally occurring conformations, and this usually determines the class to which the antibody belongs.

[0259] A portion of the CAR of the present invention, comprising an antibody or an antibody fragment thereof, may exist in various forms, in which case the antigen-binding domain is expressed as part of a continuous polypeptide chain, such as a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific 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. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment containing an scFv.

[0260] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules of their naturally occurring conformations, and this usually determines the class to which the antibody belongs.

[0261] The term "antibody light chain" refers to the smaller of two types of polypeptide chains present in antibody molecules of their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two main antibody light chain isotypes.

[0262] The term "recombinant antibody" refers to antibodies produced using recombinant DNA technology, such as antibodies expressed by bacteriophages or yeast expression systems. This term is also interpreted to mean antibodies produced by the synthesis of a DNA molecule encoding an antibody (which expresses an antibody protein) or an amino acid sequence specifying the antibody, in which case the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technologies available and well known in the industry.

[0263] The terms “antigen” or “Ag” refer to a molecule that elicits an immune response. This immune response may include either antibody production or activation of specific immune cells, or both. Those skilled in the art will expect to understand that virtually any macromolecule, encompassing all proteins or peptides, can serve as an antigen. Furthermore, antigens may be recombinant or derived from genomic DNA. Those skilled in the art will expect to understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will consequently encode an “antigen” in the same way as the term “antigen” is used herein. Furthermore, those skilled in the art will expect to understand that antigens are not necessarily encoded only by the full-length nucleotide sequence of a gene. The present invention is not limited to, but will include the use of partial nucleotide sequences of one or more genes, and it will be readily understood that these nucleotide sequences may be arranged in various combinations so as to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will expect to understand that antigens do not necessarily have to be encoded by a “gene.” It will be readily understood that antigens may be generated or synthesized, or derived from biological samples, or may be macromolecules other than polypeptides. Examples of such biological samples include, but are not limited to, tissue samples, tumor samples, and fluids containing cells or other biological components.

[0264] The terms “competitive” or “cross-competitive” are used synonymously herein to refer to the ability of an antibody molecule, such as an anti-CD20 or CD22 antibody molecule provided herein, to interfere with the binding of that antibody molecule to a target, such as human CD20 or CD22. Interference with binding may be direct or indirect (e.g., by allosteric modification of the antibody molecule or target). The extent to which an antibody molecule can interfere with the binding of another antibody molecule to its target, and therefore whether it can be said to be competitive, can be determined using competitive binding assays, such as those described herein. In some embodiments, the competitive binding assay is a quantitative competitive assay. In some embodiments, in a competitive binding assay (e.g., a competitive assay as described herein), if the binding of the first antibody molecule to the target is reduced by 10% or more, for example, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more, the first antibody molecule is said to compete with the second antibody molecule for binding to the target.

[0265] As used herein, the term “epitope” refers to a component of an antigen (e.g., human CD20 or CD22) that specifically interacts with an antibody molecule. Such components are referred herein to as epitope determinants and typically include, or are part of, elements such as amino acid side chains or sugar side chains. Epitope determinants can be defined, for example, by methods known in the art or disclosed herein, for example, by crystallography or by hydrogen-deuterium exchange. At least one or more components on the antibody molecule that specifically interact with an epitope determinant are typically located within the CDR. Typically, epitopes have specific three-dimensional structural features. Typically, epitopes have specific charge features. Some epitopes are linear epitopes, while others are conformational epitopes.

[0266] The term “anti-cancer effect” refers to a biological effect that can be demonstrated by various means, including, but is not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in expected lifespan, a decrease in cancer cell proliferation, a decrease in cancer cell survival, or an improvement in various physiological symptoms associated with the cancerous state. “Anti-cancer effect” can also be demonstrated by the ability of the peptides, polynucleotides, cells, and antibodies described herein to prevent cancer development at first site. The term “antitumor effect” refers to a biological effect that can be demonstrated by various means, including, but is not limited to, a reduction in tumor volume, a reduction in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

[0267] The term "self" refers to all material originating from the same individual from which it will later be reintroduced.

[0268] The term "allogeneic" refers to any material originating from different animals of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic if they do not have identical genes at one or more loci. In some embodiments, allogeneic material from individuals of the same species may be genetically different to a degree sufficient to interact antigenically.

[0269] The term "heterogeneous" refers to a graft derived from an animal of a different species.

[0270] The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers described herein, but not limited to, include 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 related cancers. The terms "tumor" and "cancer" are used synonymously herein, and for example, both terms encompass solid and liquid tumors, such as diffuse or circulating tumors. The terms "cancer" or "tumor," as used herein, encompass precancerous, as well as malignant cancers and tumors.

[0271] The terms “cancer-associated antigen,” “tumor antigen,” “proliferative disorder antigen,” or “antigen associated with proliferative disorder” synonymously refer to molecules (typically proteins, carbohydrates, or lipids) that are preferentially expressed on the surface of cancer cells compared to normal cells, either as a whole or as fragments (e.g., MHC / peptides), and are useful for preferential targeting of pharmacologic substances to cancer cells. In some embodiments, the tumor antigen is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In certain embodiments, the tumor antigen of the present invention is derived from cancer, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, e.g., breast cancer, prostate cancer, ovarian cancer, pancreatic cancer and similar. In some embodiments, the tumor antigen is an antigen common to a particular proliferative disorder. In some embodiments, cancer-associated antigens are cell surface molecules that are overexpressed in cancer cells compared to normal cells, e.g., 1x expression, 2x overexpression, 3x overexpression, or more compared to normal cells. In some embodiments, cancer-associated antigens are cell surface molecules that are improperly synthesized in cancer cells, e.g., molecules containing deletions, additions, or mutations compared to molecules expressed in normal cells. In some embodiments, cancer-associated antigens are expressed exclusively on the cell surface of cancer cells, either whole or as fragments (e.g., MHC / peptide), and are neither synthesized nor expressed on the surface of normal cells. In some embodiments, the CAR of the present invention comprises a CAR containing an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to a peptide presented by MHC. Typically, peptides derived from endogenous proteins fill the pocket of a major histocompatibility complex (MHC) class I molecule and are recognized by the T cell receptor (TCR) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a distinct class of cell surface targets for immunotherapy.TCR-like antibodies targeting viral or tumor antigen-derived peptides in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described [see, for example, Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Bood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100]. For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage-presenting libraries.

[0272] The phrase “diseases associated with CD19 expression” includes, for example, proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as spinal malformations, myelodysplastic syndromes, or preleukemic states, or conditions related to cells that express CD19 (e.g., wild-type or mutated CD19) or that have expressed it at any given time; or non-cancerous signs related to cells that express CD19. To avoid misunderstanding, diseases associated with CD19 expression include, for example, conditions related to cells that previously expressed CD19 but do not currently express it because CD19 expression is downregulated due to treatment with a CD19-targeting molecule, e.g., CD19 CAR. In one embodiment, cancers associated with CD19 expression are hematological cancers. In one embodiment, hematological cancers are leukemia or lymphoma. In one embodiment, cancers associated with CD19 expression include, but are not limited to, one or more acute leukemias such as B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), and acute lymphoblastic leukemia (ALL); and cancers and malignancies such as, but are not limited to, one or more chronic leukemias such as chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Additional cancerous or hematological conditions associated with CD19 expression include, but are not limited to, B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms, 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 (MCL), marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström macroglobulinemia, and “preleukemic conditions,” a diverse collection of hematological conditions associated with the ineffective production (or dysplasia) of blood cells in the bone marrow, and similar conditions.Furthermore, diseases associated with CD19 expression include, but are not limited to, atypical and / or non-typical cancers, malignancies, precancerous conditions, or proliferative disorders associated with CD19 expression. Non-cancer-related conditions associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, CD19-expressing cells express CD19 mRNA or have expressed it at some point in time. In some embodiments, CD19-expressing cells produce CD19 protein (e.g., wild-type or mutant), and this CD19 protein may be present at normal or reduced levels. In some embodiments, CD19-expressing cells produced detectable levels of CD19 protein at some point in time, but subsequently did not produce substantially detectable levels of CD19 protein.

[0273] The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antibody fragment of the present invention by standard techniques known in the industry, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the industry. 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). Therefore, one or more amino acid residues in the CAR of the present invention may be replaced with other amino acid residues of the same side chain family, and the modified CAR can be tested using the functional assays described herein.

[0274] The term "stimulus" refers to the primary response induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex or CAR) to its cognate ligand (or, in the case of a CAR, a tumor antigen), thereby mediating a signaling event, such as, but not limited to, signaling via the TCR / CD3 complex or signaling via the appropriate NK receptor or signaling domain of a CAR. Stimuli may also mediate the altered expression of a particular molecule.

[0275] The term “stimulating molecule” refers to a molecule expressed by immune cells, such as T cells, NK cells, or B cells, that gives rise to an intracellular signaling sequence that modulates the activation of immune cells in the form of a stimulus, with respect to at least some aspects of the immune cell signaling pathway. In one aspect, the signal is a primary signal initiated, for example, by the binding of a TCR / CD3 complex to an MHC molecule presenting a peptide, thereby mediating T cell responses such as proliferation, activation, differentiation, and homogeneous reactions, but not limited to these. The primary intracellular signaling sequence acting in the form of a stimulus (also referred to as the “primary signaling domain”) may contain a signaling motif, which is known as an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing intracellular signaling sequences having a specific use in the present invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In certain CARs of the present invention, the intracellular signaling domain in any one or more CARs of the present invention comprises an intracellular signaling sequence, for example, the primary signaling sequence of CD3-zeta. In certain CARs of the present invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 17, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, and their counterparts. In certain CARs of the present invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 43, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, and their counterparts.

[0276] The term "antigen-presenting cell" or "APC" refers to immune system cells, such as accessory cells (e.g., B cells, dendritic cells, and allogenes), that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and cause T cells to present them.

[0277] When used herein, “immune effector cells” refer to cells involved in promoting an immune response, such as an immune effector response. Examples of immune effector cells include T cells, such as alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NK-T) cells, mast cells, and myeloid phagocytic cells.

[0278] When this term is used herein, “immune effector function or immune effector response” refers, for example, to the function or response of immune effector cells that enhance or promote the immune attack of target cells. For example, immune effector function or response refers to the properties of T or NK cells that promote the killing or inhibition of the growth or proliferation of target cells. In the case of T cells, primary and secondary stimuli are examples of immune effector function or response.

[0279] The term "effector function" refers to a specialized function of a cell. The effector function of a T cell could be, for example, helper activity such as cytolytic activity or cytokine secretion.

[0280] When this term is used herein, the “intracellular signaling domain” refers to the intracellular portion of a molecule. Intracellular signaling domains can generate signals that promote the immune effector functions of cells containing CARs, such as CART cells. Examples of immune effector functions in CART cells include cytolytic activity and helper activity, such as cytokine secretion. In embodiments, the intracellular signaling domain is a portion of a protein that transmits effector function signals, causing cells to perform specialized functions. While the entire intracellular signaling domain may be employed, it is often not necessary to use the entire chain. As long as a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, provided that it transmits the effector function signal. Therefore, the term intracellular signaling domain encompasses any truncated portion of an intracellular signaling domain sufficient to transmit an effector function signal.

[0281] In some embodiments, the intracellular signaling domain may include a primary intracellular signaling domain. Typical primary intracellular signaling domains include those derived from molecules involved in primary stimulation or antigen-dependent simulation. In some embodiments, the intracellular signaling domain may include a co-stimulatory intracellular domain. Typical co-stimulatory intracellular signaling domains include those derived from molecules involved in co-stimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0282] The primary intracellular signaling domain may contain an immune receptor tyrosine-based activation motif or a signaling motif known as an ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, CD32, DAP10, and DAP12.

[0283] The terms “zeta” or, in lieu of “zeta chain,” “CD3-zeta,” or “TCR-zeta” are defined as the protein provided under GenBan accession number BAG36664.1, or equivalent residues from non-human species, e.g., mice, rodents, monkeys, apes, and their allies. The “zeta-stimulating domain” or, in lieu of “CD3-zeta-stimulating domain” or “TCR-zeta-stimulating domain” are defined as amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, sufficient to functionally transmit the initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of zeta comprises residues 52 to 164 of GenBank accession number BAG36664.1, or equivalent residues from non-human species, e.g., mice, rodents, monkeys, apes, and their allies that are functional orthologs. In one embodiment, the “zeta-stimulating domain” or “CD3-zeta-stimulating domain” is the sequence provided under Sequence ID No. 17. In one embodiment, the "zeta-stimulating domain" or "CD3-zeta-stimulating domain" is the sequence provided as Sequence ID No. 43.

[0284] The term "costimulatory molecule" refers to a co-stimulatory partner on a T cell that mediates a T cell costimulatory response, such as proliferation, by specifically binding to a costimulatory ligand, although these molecules are not limited to costimulatory ligands. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Examples of costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1B B(CD137), B7-H3, CDS, ICAM-1, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, IT GA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2 , CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), Ligands that specifically bind to CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83 are examples.

[0285] The co-stimulatory intracellular signaling domain refers to the intracellular portion of the co-stimulatory molecule. The intracellular signaling domain may include the entire intracellular portion, the entire native intracellular signaling domain of the molecule from which it is obtained, or functional fragments or derivatives thereof.

[0286] The term "4-1BB" refers to a member of the TNFR superfamily having the sequence amino acid sequence provided as GenBank accession number AAA62478.2, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes and their relatives, and the "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes and their relatives. In one embodiment, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 16 or equivalent residues from non-human species, such as mice, rodents, monkeys, apes and their relatives.

[0287] The term “coding” refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a specific sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a specific sequence of amino acids, and the biological properties that result from it, serving as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, in cells or other biological systems, when proteins are produced by the transcription and translation of mRNA corresponding to a gene, that gene, cDNA, or RNA codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually shown in sequence listings, and the non-coding strand used as a template for the transcription of a gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0288] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate of each other or that encode the same amino acid sequence. Furthermore, the phrase "nucleotide sequence encoding a protein or RNA" may, in some cases, contain introns to the extent that the nucleotide sequence encoding that protein may contain introns.

[0289] The terms “effective dose” or “therapeutic dose” are used synonymously herein and refer to the amount of a compound, preparation, material, or composition, as described herein, that is effective in achieving a particular biological outcome.

[0290] The term "endogenous" refers to any material that originates from an organism, cell, tissue, or system, or any material produced within them.

[0291] The term "exogenous" refers to any material introduced from outside an organism, cell, tissue, or system, or any material produced outside of them.

[0292] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0293] The term "transfer vector" refers to a composition containing isolated nucleic acids that can be used to deliver isolated nucleic acids into cells. Numerous vectors are known in the industry, but are not limited to, linear polynucleotides, polynucleotides conjugated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "transfer vector" encompasses autonomously replicating plasmids or viruses. This term should also be interpreted to further encompass non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and analogues. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, and analogues.

[0294] The term “expression vector” refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence ligated to the nucleotide sequence to be expressed in a functional manner. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be supplied by the host cell or within the in vitro expression system. Expression vectors encompass everything known in the art, including, for example, cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0295] The term "lentivirus" refers to a genus of retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and because they can deliver a significant amount of genetic information to the host cell's DNA, they are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0296] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, particularly self-inactivating lentiviral vectors, such as those described in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used in clinical settings include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX® vector system, and similar products. Non-clinical lentiviral vectors are also available and are expected to be known to those skilled in the art.

[0297] The terms "homologous" or "identical" refer to the identity of subunit sequences between two macromolecules, for example between two nucleic acid molecules, for example between two DNA molecules or two RNA molecules, or between two polypeptide molecules. If the positions of subunits in both molecules are occupied by the same monomer subunits, for example, if the positions in each of two DNA molecules are occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a linear function of the number of matched or homologous positions. For example, if half of the positions in the two sequences are homologous (e.g., 5 positions out of 10 subunits in a polymer), then the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0298] The “humanized” form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof [e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody] containing the smallest sequence derived from the non-human immunoglobulin. In most cases, the humanized antibody and its antibody fragments are human immunoglobulins (recipient antibodies or antibody fragments) in which residues from the recipient’s complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, that possess the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody / antibody fragment may contain residues not found in either the recipient antibody or the transferred CDR or framework sequence. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Generally, humanized antibodies or their antibody fragments are expected to contain at least one, typically two, substantially all of variable domains, where all or substantially all of the CDR region corresponds to the CDR region of a non-human immunoglobulin, and all or a substantial portion of the FR region is the FR region of a human immunoglobulin sequence. Humanized antibodies or antibody fragments may also contain at least a portion of the immunoglobulin constant region (Fc), typically the immunoglobulin constant region (Fc) 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.

[0299] "Completely human" refers to immunoglobulins, such as antibodies or antibody fragments, where the entire molecule is of human origin or consists of an amino acid sequence identical to that of the human form of an antibody or immunoglobulin.

[0300] The term "isolated" means that something has been altered or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is "not isolated," but the same nucleic acid or peptide is "isolated" if it has been partially or completely separated from the material that coexists with it in its natural state. Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0301] In the present invention, the following abbreviations for commonly existing nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0302] The terms "functionally linked" or "transcriptional regulation" refer to the functional linking of a regulatory sequence to a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, if a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence is functionally linked to the second nucleic acid sequence. For example, if a promoter influences the transcription or expression of a coding sequence, the promoter is functionally linked to the coding sequence. Functionally linked DNA sequences may be contiguous with each other, or they may be within the same reading frame, for example, if it is necessary to fused the coding regions of two proteins.

[0303] The term "parenteral" administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, intratumoral, or infusion techniques.

[0304] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof in either single-stranded or double-stranded forms. The term “nucleic acid” encompasses genes, cDNA, or mRNA. In one embodiment, a nucleic acid molecule is a synthetic (e.g., chemically synthesized) nucleic acid molecule or a recombinant nucleic acid molecule. Unless otherwise specified, the term encompasses nucleic acids containing analogs or derivatives of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also substantially encompasses its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, in addition to sequences explicitly presented. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a hybrid base and / or deoxyinosine residue [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. Probes 8:91-98 (1994)].

[0305] The terms “peptide,” “polypeptide,” and “protein” are used synonymously and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide encompasses any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, this term refers to both short chains, commonly referred to in the industry as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the industry as proteins, of which many types exist. Examples of “polypeptides” include, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Examples of polypeptides include native peptides, recombinant peptides, or combinations thereof.

[0306] The term "promoter" refers to a DNA sequence recognized by a cellular synthetic mechanism, or the introduced synthetic mechanism, that is necessary to initiate the specific transcription of a polynucleotide sequence.

[0307] The term "promoter / regulatory sequence" refers to a nucleic acid sequence necessary for the expression of a gene product that is ligated to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also contain enhancer sequences and other regulatory factors necessary for the expression of the gene product. A promoter / regulatory sequence may, for example, be a promoter / regulatory sequence that expresses a gene product in a tissue-specific manner.

[0308] The term “constitutive” promoter refers to a nucleotide sequence that, when linked to a polynucleotide that codes for or designates a gene product, causes the cell to produce that gene product under most or all physiological conditions.

[0309] The term "inducible" promoter refers to a nucleotide sequence that, when linked to a polynucleotide that codes for or designates a gene product, causes a cell to produce a gene product only when an inducer, essentially corresponding to the promoter, is present in the cell.

[0310] The term "tissue-specific" promoter refers to a nucleotide sequence that, when linked to a polynucleotide that codes for a gene or is specified by a gene, causes the cell to produce a gene product only when the cell is substantially the tissue type corresponding to the promoter.

[0311] The term “flexible polypeptide linker” or “linker,” when used in the context of scFv, refers to a peptide linker consisting of amino acids such as glycine and / or serine residues, used alone or in combination to link a variable heavy chain region and a variable light chain region together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker with an amino acid sequence (Gly-Gly-Gly-Ser) n The formula includes, 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 and n=10 (SEQ ID NO: 105). In one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 (SEQ ID NO: 106) or (Gly4Ser)3 (SEQ ID NO: 107). In another embodiment, the linker includes multiple iterations of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 108). The linkers described in WO2012 / 138475, incorporated herein by reference, are also included within the scope of the present invention.

[0312] 5' cap (RNA cap, RNA7-methylguanosine cap or RNAm 7A 5' cap (also known as a G-cap), as used herein, is a modified guanine nucleotide added to the “prefix” or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of terminal groups that are linked to the first nucleotide to be transcribed. Its presence is important for ribosome recognition and protection from RNases. Capping is performed by coupling with transcription, with each process transcribing together in a manner that influences the other. Immediately after transcription initiation, the 5' end of the synthesized mRNA is bound to a complex that synthesizes the cap, associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions necessary for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping components may be modified to regulate the functionality of the mRNA, such as its stability or translation efficiency.

[0313] As used herein, "in vitro transcribed RNA" refers to RNA synthesized in vitro, such as mRNA. Generally, in vitro transcribed RNA is produced from an in vitro transcription vector. An in vitro transcription vector contains a template used to produce in vitro transcribed RNA.

[0314] As used herein, "poly(A)" refers to a series of adenosines attached to mRNA by polyadenylation. In some embodiments of the construct for transient expression, the number of poly(A) sequences is between 50 and 5000 (SEQ ID NO: 28), e.g., more than 64, e.g., more than 100, e.g., more than 300 or 400. The poly(A) sequence may be chemically or enzymatically modified to modulate mRNA functionality such as localization, stability, or translation efficiency.

[0315] As used herein, "polyadenylation" refers to the covalent linking of a polyadenylyl component or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' end. The 3' poly(A) tail is a long sequence (often hundreds) of adenine nucleotides added to pre-mRNA via the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to a transcript containing a specific sequence, the polyadenylation signal. The poly(A) tail and the protein it binds to help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA transport out of the cell nucleus, and translation. Polyadenylation occurs in the cell nucleus immediately after DNA transcription to RNA, and can also occur later in the cytoplasm. After transcription is complete, the mRNA strand is cleaved via an endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end at the cleavage site.

[0316] As used herein, "transient" refers to the expression of an unintegrated transgene over a period of several hours, days, or weeks, where the expression period is shorter than that of a gene integrated into the genome or contained within a stable plasmid replicon in a host cell.

[0317] The term "signaling pathway" refers to the biochemical relationships between various signaling molecules that play a role in signal transmission from one part of a cell to another. The phrase "cell surface receptor" encompasses molecules and molecular complexes that can receive signals and cross the cell membrane to transmit those signals.

[0318] The term "target" is intended to encompass organisms capable of eliciting an immune response (e.g., mammals, humans).

[0319] The term "substantially purified" refers to cells that essentially contain no other cell types. It can also refer to cells isolated from other cell types that normally coexist in their natural environment. In some cases, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells isolated from cells that naturally coexist in their natural state. In some embodiments, such cells are cultured in vitro. In other embodiments, such cells are not cultured in vitro.

[0320] The term "treatment," as used herein, means a procedure. Therapeutic effects are obtained by recovery, suppression, remission, or eradication of a disease.

[0321] As used herein, the term "prevention" means the prevention or preventive action of a disease or medical condition.

[0322] In the context of this invention, "tumor antigen," "hyperproliferative disorder antigen," or "antigen associated with hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder. In certain embodiments, the hyperproliferative disorder antigens of this invention are, but are not limited to, cancer-derived, such as primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and similar cancers.

[0323] The term "transfection," "transformation," or "transduction" refers to the process of introducing or transferring exogenous nucleic acids into host cells. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acids. The cells include the primary cells and their descendants.

[0324] A subject "responds" to treatment if the parameters of cancer in a subject (e.g., hematological cancer, e.g., cancer cell growth, proliferation, and / or survival) are delayed or reduced by a detectable amount, e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, as determined by any appropriate measure, e.g., mass, cell count, or volume. In one example, a subject responds to treatment if they experience an extended expected lifespan by more than about 5%, 10%, 20%, 30%, 40%, or 50% compared to the expected lifespan predicted without treatment. In another example, a subject responds to treatment if they have increased disease-free survival, overall survival, or increased progression-free survival. Several methods can be used to determine whether a patient responds to treatment, including the criteria provided by the NCCN Clinical Practice Guidelines for Oncology [NCCN Guidelines®]. For example, in the context of B-ALL, a complete response or complete response case may include one or more of the following: <5% BM blasts, >1000 neutrophils / ANC( / μL); >100,000 platelets( / μL) with no circulating blasts or extramedullary disease (no lymphadenopathy, megasplenia, cutaneous / gingival infiltration / testicular mass / CNS lesions), tricellular hematopoiesis, and no relapse over 4 weeks. A partial response case may include one or more of the following: >50% reduction in BM blasts, >1000 neutrophils / ANC( / μL); >100,000 platelets( / μL). A non-response case may show disease progression, e.g., >25% in terms of BM blasts.

[0325] As used herein, "refractory" refers to a disease, such as cancer, that does not respond to treatment. In some embodiments, a refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. Refractory cancer is also called resistant cancer.

[0326] The term “relapse,” as used herein, refers to the reappearance of cancer after the initial response period (e.g., complete or partial response). The initial response period may include a decrease in the level of cancer cells to below a certain threshold, e.g., 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance may include an increase in the level of cancer cells to above a certain threshold, e.g., 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, reappearance may include, for example, the reappearance of blasts in the blood, bone marrow (>5%), or any extramedullary location after a complete response. A complete response in this context may include <5% BM blasts. More generally, in some embodiments, a response (e.g., complete or partial response) may include the absence of detectable MRD (minimal residual disease). In one embodiment, the initial response period lasts at least 1, 2, 3, 4, 5, or 6 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 6, 8, 10, or 12 months, or at least 1, 2, 3, 4, or 5 years.

[0327] In some embodiments, therapies including CD19 inhibitors, such as CD19 CAR therapy, may result in relapse or resistance to treatment. Relapse or resistance may be caused by CD19 deficiency (e.g., antigen deficiency mutation) or by other CD19 alterations that reduce CD19 levels (e.g., caused by clonal selection of CD19-negative clones). Cancers having such CD19 deficiency or alteration are referred to herein as “CD19-negative cancer” or “CD19-negative recurrent cancer.” It is understood that CD19-negative cancer must not be a 100% deficiency of CD19, but rather have a reduction sufficient to reduce the effectiveness of CD19 therapy to the point where the cancer relapses or becomes refractory. In some embodiments, CD19-negative cancer results from CD19 CAR therapy.

[0328] The term "specifically binding" refers to an antibody or ligand that recognizes and binds to a binding partner protein (e.g., a stimulating tumor antigen) present in the sample, but these antibodies or ligands do not substantially recognize or bind to other molecules in the sample.

[0329] As used herein, the term “pharmaceutically acceptable salt” means a salt that, within the bounds of sound medical judgment, is suitable for use in contact with the target tissue, is free from excessive toxicity, irritation, allergic reactions and similar effects, and is commensurate with a reasonable cost-benefit ratio. pharmaceutically acceptable salts are well known in the industry. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19.

[0330] When this term is used herein, a “modulated chimeric antigen receptor (RCAR)” refers to a set of polypeptides, typically two polypeptide sets in its simplest embodiment, that, when present in RCARX cells, can bring to those RCARX cells specificity to target cells, typically cancer cells, and the generation or proliferation of modulated intracellular signals, thereby optimizing the immunoeffector properties of those RCARX cells. RCARX cells rely at least partially on the antigen-binding domain to provide specificity to target cells containing antigens bound by the antigen-binding domain. In some embodiments, the RCAR incorporates a dimerization switch that can couple the intracellular signaling domain to the antigen-binding domain in the presence of a dimerization molecule.

[0331] When this term is used herein, “membrane anchor” or “membrane anchoring domain” refers to a polypeptide or moiety sufficient to fix an extracellular or intracellular domain to the plasma membrane, such as a myristoyl group.

[0332] When this term is used herein, for example in relation to RCAR, a “switch domain” refers to an object, typically a polypeptide-based object, that associates with another switch domain in the presence of a dimerizing molecule. The association results in a functional coupling between a first object, for example, a fused first object, linked to the first switch domain, and a second object, for example, a fused second object, linked to the second switch domain. The first and second switch domains are collectively referred to as a dimerizing switch. In embodiments, the first and second switch domains are identical to each other, for example, polypeptides having the same primary amino acid sequence, and are collectively referred to as a homodimerizing switch. In embodiments, the first and second switch domains are different to each other, for example, polypeptides having different primary amino acid sequences, and are collectively referred to as a heterodimerizing switch. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based object, for example, an FKBP or FRB-based object, and the dimerizing molecule is a small molecule, for example, a rapalogue. In one embodiment, the switch domain is an scFv that binds to a polypeptide-based object, such as a myc peptide, and the dimerizing molecule is a polypeptide, a fragment thereof, or a polypeptide polymer, such as a myc ligand or a polymer of myc ligands that binds to one or more myc scFvs. In another embodiment, the switch domain is a polypeptide-based object, such as a myc receptor, and the dimerizing molecule is an antibody or a fragment thereof, such as a myc antibody.

[0333] When this term is used herein, for example in reference to RCAR, “dimerizing molecule” refers to a molecule that facilitates the association of the first switch domain and the second switch domain. In embodiments, the dimerizing molecule is either not naturally present in the subject or is not present at concentrations expected to cause significant dimerization. In embodiments, the dimerizing molecule is a small molecule, such as rapamycin or rapalog, for example, RAD001.

[0334] The term “low dose for immune enhancement” refers to a dose of an mTOR inhibitor that partially, but not completely, inhibits mTOR activity, as measured, for example by P70S6 kinase activity inhibition, when used in conjunction with an mTOR inhibitor, such as an allosteric mTOR inhibitor, such as RAD001 or rapamycin, or a catalytic mTOR inhibitor. For example, a method for evaluating mTOR activity by P70S6 kinase inhibition is discussed herein. This dose is insufficient to cause complete immunosuppression but sufficient to enhance the immune response. In one embodiment, a low dose of immune enhancement mTOR inhibitor causes a decrease in the number of PD-1 positive T cells and / or an increase in the number of PD-1 negative T cells, or an increase in the PD-1 negative T cell / PD-1 positive T cell ratio. In one embodiment, a low dose of immune enhancement mTOR inhibitor causes an increase in the number of naive T cells. In one embodiment, a low dose of immune enhancement mTOR inhibitor is as follows: For example, the following marker in memory T cells, for example in memory T cell precursors: CD62L high CD127 high CD27 + , and increased expression of one or more BCL2; For example, decreased KLRG1 expression in memory T cells, for example, in memory T cell precursors; and Memory T cell precursors, for example, have the following characteristics: CD62L high Increase, CD127 high Increase, CD27 +Increased number of cells having one or a combination of the following: increased KLRG1, decreased KLRG1, and increased BCL2. This causes one or more of the changes described above, and any of these changes occur, for example, compared to an untreated subject, at least temporarily.

[0335] Scope: Throughout this disclosure, various aspects of the invention may be indicated in the form of scope. It is understood that such indications are for convenience and brevity only and should not be construed as inflexible limitations on the scope of the invention. Accordingly, scope indications shall be deemed to specifically disclose all possible subranges, in addition to the individual numerical values ​​within those ranges. For example, a scope indication such as 1 to 6 is expected to be deemed to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, in addition to the individual numerical values ​​within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity encompasses something that has 95%, 96%, 97%, 98%, or 99% identity, and further encompasses subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the range.

[0336] explanation CD19 inhibitor and binding domain Compositions and methods of use of a CD19 chimeric antigen receptor (CAR) for treating diseases such as cancer are provided herein. The methods include, among other things, administering the CD19 CAR described herein in combination with another agent, such as a B-cell inhibitor. The methods also include administering the CD19 CAR described herein to treat lymphomas, such as Hodgkin lymphoma.

[0337] In one embodiment, the present invention provides a number of chimeric antigen receptors (CARs) comprising an antibody or antibody fragment processed to specifically bind to the CD19 protein. In one embodiment, the present invention provides cells (e.g., T cells) processed to express a CAR, where the CAR T cells ("CART") exhibit anti-cancer properties. In one embodiment, cells are transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, cells (e.g., T cells) are 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, cells can stably express a CAR. In another embodiment, cells (e.g., T cells) are transfected with a nucleic acid encoding a CAR, such as mRNA, cDNA, or DNA. In some such embodiments, cells can transiently express a CAR.

[0338] In one embodiment, the anti-CD19 protein-binding portion of the CAR is an scFv antibody fragment. In one embodiment, such antibody fragments are functional in that they retain equivalent binding affinity, for example, they bind to the same antigen with equivalent affinity to the IgG antibody from which they originated. In one embodiment, such antibody fragments are functional in that they produce biological responses such as activation of an immune response, inhibition of signal initiation from its target antigen, inhibition of kinase activity, and similar responses, which are expected to be understood by those skilled in the art, but are not limited to these. In one embodiment, the CD19 antigen-binding domain of the CAR is a humanized scFv antibody fragment compared to the mouse sequence of its origin. In one embodiment, the parental mouse scFv sequence is the CAR19 construct provided in PCT Publication WO2012 / 079000 and provided herein as Sequence ID No. 59. In one embodiment, the anti-CD19-binding domain is the scFv described in WO2012 / 079000 and provided as Sequence ID No. 59, or a sequence that is at least 95%, e.g., 95-99%, identical thereto. In one embodiment, the anti-CD19 binding domain is a portion of the CAR construct provided in PCT Publication WO2012 / 079000 and provided herein by SEQ ID NO: 58, or a sequence that is at least 95%, e.g., 95% to 99%, identical thereto. In one embodiment, the anti-CD19 binding domain comprises at least one (e.g., two, three, four, five, or six) CDRs selected from Table 4 and / or Table 5.

[0339] In some embodiments, the antibody of the present invention is incorporated into a chimeric antigen receptor (CAR). In one embodiment, the CAR comprises a 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 substituted with one or more sequences selected from SEQ ID NOs: 1 to 12. In one embodiment, the scFv domains of SEQ ID NOs: 1 to 12 are humanized variants of the scFv domain of SEQ ID NO: 59, which are mouse-derived scFv fragments that specifically bind to human CD19. This humanization of the mouse scFv may be desirable for clinical conditions in patients undergoing CART19 treatment, such as treatment with T cells transduced with a CAR19 construct, where mouse-specific residues can induce a human anti-mouse antigen (HAMA) response.

[0340] In one embodiment, the anti-CD19 binding domain, for example, the humanized scFv which is part of the CAR of the present invention, is encoded by a transgene whose codons in its sequence are optimized for expression in mammalian cells. In one embodiment, the entire CAR construct of the present invention is encoded by a transgene whose codons in its entire sequence are optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased across different species. Such codon degeneracy makes it possible to code for the same polypeptide with various nucleotide sequences. Various codon optimization methods are known in the art, including, for example, those disclosed in at least U.S. Patents 5,786,464 and 6,114,148.

[0341] In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 1. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 2. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 3. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 4. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 5. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 6. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 7. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 8. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 9. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 10. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 11. In one embodiment, the humanized CAR19 includes the scFv portion shown in SEQ ID NO: 12.

[0342] In one embodiment, the CAR of the present invention combines a specific antibody-antigen binding domain with an intracellular signaling molecule. For example, in some embodiments, the intracellular signaling molecule may include, but is not limited to, the CD3-zeta chain, 4-1BB and CD28 signaling modules, and combinations thereof. In one embodiment, the CD19 CAR comprises a CAR selected from the sequences provided in one or more of SEQ ID NOs: 31-42. In one embodiment, the CD19 CAR comprises the sequence provided in SEQ ID NO: 31. In one embodiment, the CD19 CAR comprises the sequence provided in SEQ ID NO: 32. In one embodiment, the CD19 CAR comprises the sequence provided in SEQ ID NO: 33. In one embodiment, the CD19 CAR comprises the sequence provided in SEQ ID NO: 34. In one embodiment, the CD19 CAR comprises the sequence provided in SEQ ID NO: 35. In one embodiment, the CD19 CAR comprises the sequence provided in SEQ ID NO: 36. In ...

Claims

1. Cells expressing a CD19-binding CAR molecule, such as CD19CAR, for use in the treatment of subjects having a disease related to CD19 expression, wherein the subject has received, is receiving, or is scheduled to receive one or more B cell inhibitors, the B cell inhibitor comprising one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

2. A method for treating a subject having a disease related to CD19 expression, comprising administering to the subject an effective number of one or more cells expressing a CD19-binding CAR molecule, for example, CD19CAR, in combination with one or more B-cell inhibitors, wherein the B-cell inhibitors include one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

3. CD19 inhibitors, for example, one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a, for use in the treatment of subjects that are unresponsive, partially responsive or relapsed to CD19 CAR therapy, or identified as such.

4. A method for treating a patient who is unresponsive, partially responsive, or relapsed to CD19 inhibitor therapy, for example, CD19 CAR therapy, or who is identified as such, comprising administering to the patient one or more B cell inhibitors, for example, inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

5. A composition for use or method according to any one of claims 1 to 4, wherein the B cell inhibitor comprises one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1.

6. A composition for use or method according to any one of claims 1 to 5, wherein the B cell inhibitor comprises an effective number of one or more cells expressing a CAR molecule bound to one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

7. A composition for use or method according to any one of claims 1 to 6, wherein one or more cells expressing a CAR molecule bound to CD19 are administered simultaneously with, before or after, the one or more B cell inhibitors.

8. A composition for use or method according to any one of claims 1 to 7, wherein the subject is identified as having relapsed or relapsed after treatment in one or more cells expressing a CD19-binding CAR molecule, for example, CD19 CAR.

9. The subject is identified as having relapsed or having relapsed based on the reappearance of one or more blast cells in the blood, bone marrow (>5%) or any extramedullary site after a complete response, or Based on the detection of CD19-blast cells at a level higher than a predetermined threshold, for example, 1%, 2%, 3%, 4%, 5%, or 10%, the subject is identified as having relapsed or as having relapsed. The subject is identified as having relapsed or having relapsed based on an increase in the levels of one or more RNAs of MIR199A1, MIR1203, uc021ovp, ITM2C, or HLA-DQB1, or The aforementioned subjects are identified as having relapsed or having relapsed based on a decrease in the RNA levels of one or more of the following: PPIAL4D, TTTY10, TXLNG2P, MIR4650-1, KDM5D, USP9Y, PRKY, RPS4Y2, RPS4Y1, NCRNA00185, SULT1E1, and EIF1AY. A composition for use or method according to claim 11.

10. A composition for use or method according to any one of claims 1 to 9, further comprising performing lymphocyte depletion before administering, for example, one or more cells expressing a CAR molecule bound to CD19, wherein the lymphocyte depletion may include administering one or more of melphalan, cytoxane, cyclophosphamide, and fludarabine.

11. A composition for use or method according to any one of claims 1 to 10, wherein the B cell inhibitor comprises an effective number of cells expressing one or more cells expressing a CAR molecule that binds to CD10, for example, as described herein; a CAR molecule that binds to CD20, for example, as described herein; a CAR molecule that binds to CD22, for example, as described herein; a CAR molecule that binds to CD34, for example, as described herein; a CAR molecule that binds to CD123, for example, as described herein; a CAR molecule that binds to FLT-3, for example, as described herein; or a CAR molecule that binds to ROR1, for example, as described herein.

12. A composition for use or method according to any one of claims 1 to 11, wherein the CD19 CAR comprises an antibody or antibody fragment comprising a CD19-binding domain, a transmembrane domain, and an intracellular signaling domain including a stimulating domain, the CD19-binding domain comprising one or more (e.g., all three) of the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of any CD19 scFv or light chain-binding domain amino acid sequence listed in Table 2 or 3, and one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of any CD19 scFv or heavy chain-binding domain amino acid sequence listed in Table 2 or 3.

13. A composition for use or method according to any one of claims 1 to 12, wherein CD19 CAR comprises a light chain variable region of any of the scFV listed in Table 2 or 3, and a heavy chain variable region of any of the scFv listed in Table 2 or 3.

14. A composition for use or method according to any one of claims 1 to 13, wherein the CD19 CAR comprises a CD19-binding domain comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a sequence having 95-99% identity with them, or the polypeptide of SEQ ID NO:

58.

15. A composition for use or method according to any one of claims 1 to 14, wherein the B cell inhibitor comprises a CD20 CAR, the CD20 CAR comprises an antibody or antibody fragment comprising a CD20-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulating domain, the CD20-binding domain comprising one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD20 light chain-binding domain amino acid sequence listed in Table 13, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD20 heavy chain-binding domain amino acid sequence listed in Table 12A or 12B.

16. A composition for use or method according to any one of claims 1 to 15, wherein the B cell inhibitor comprises a CD22 CAR, the CD22 CAR comprises an antibody or antibody fragment comprising a CD22-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulating domain, the CD22-binding domain comprising one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD22 light chain-binding domain amino acid sequence listed in Table 8A or 8B, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD22 heavy chain-binding domain amino acid sequence listed in Table 7A, 7B, or 7C.

17. A composition for use or method according to claim 16, wherein the CD22 CAR includes any of the light chain variable regions listed in Table 10A or 10B, or any of the heavy chain variable regions listed in Table 9A or 9B, or includes any of the light chain variable regions listed in Table 10A or 10B and any of the heavy chain variable regions listed in Table 9A or 9B.

18. A composition for use or method according to any one of claims 1 to 17, wherein the B cell inhibitor comprises a CD123 CAR, the CD123 CAR comprises an antibody or antibody fragment comprising an intracellular signaling domain comprising a CD123-binding domain, a transmembrane domain, and a stimulating domain, the CD123-binding domain comprising one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD123 light chain-binding domain amino acid sequence listed in Table 18, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD123 heavy chain-binding domain amino acid sequence listed in Table 17.

19. A composition for use or method according to claim 18, wherein the CD123 CAR includes any of the light chain variable regions listed in Table 16, or any of the heavy chain variable regions listed in Table 16, or any of the light chain variable regions listed in Table 16 and any of the heavy chain variable regions listed in Table 16.

20. A composition for use or method according to any one of claims 1 to 19, wherein the B cell inhibitor comprises a CD123 CAR, the CD123 CAR comprises an antibody or antibody fragment comprising an intracellular signaling domain comprising a CD123 binding domain, a transmembrane domain, and a stimulating domain, the CD123 binding domain comprising one or more light chain complementarity determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD123 light chain binding domain amino acid sequence listed in Table 27, and one or more heavy chain complementarity determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD123 heavy chain binding domain amino acid sequence listed in Table 26.

21. A composition for use or method according to claim 20, wherein the CD123 CAR includes any of the light chain variable regions listed in Table 25, or any of the heavy chain variable regions listed in Table 25, or includes any of the light chain variable regions listed in Table 25 and any of the heavy chain variable regions listed in Table 25.

22. A composition for use or method according to any one of claims 1 to 21, wherein the B cell inhibitor comprises a CAR, the CAR comprises an antibody or antibody fragment comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulating domain, the antigen-binding domain comprising one or more (e.g., all) of light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3), and one or more (e.g., all) of heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3).

23. A composition for use or method according to any one of claims 1 to 22, wherein the B cell inhibitor comprises a CAR containing scFv.

24. A composition for use or method according to any one of claims 1 to 23, wherein the B cell inhibitor comprises a CAR including a transmembrane domain, the transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

25. The composition for use or method according to claim 22, wherein the antigen-binding domain is connected to the transmembrane domain by a hinge region, and the hinge region may include sequence number 14 or a sequence having 95-99% identity therewith.

26. The composition for use or method according to claim 22, wherein the co-stimulatory domain is a 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), and the co-stimulatory domain may include the sequence of SEQ ID NO: 16 or SEQ ID NO:

51.

27. The intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 zeta; or The intracellular signaling domain includes the sequence of SEQ ID NO: 16 and / or the sequence of SEQ ID NO: 17 or SEQ ID NO:

43. A composition for use or method according to claim 22.

28. The composition for use or method according to claim 22, wherein the CAR further comprises a leader sequence, and the leader sequence may comprise sequence number 13.

29. A composition for use or method according to any one of claims 1 to 28, wherein the cells include T cells or NK cells.

30. The disease associated with CD19 expression is selected from proliferative disorders such as cancer, tumors or malignant tumors, or precancerous conditions such as spinal malformations, myelodysplastic syndromes or preleukemic states, or is a non-cancer-related sign associated with CD19 expression; or The disease is one or more of the following: blood cancer, acute leukemia, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoblastic leukemia (ALL), chronic leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, or myeloma; or, The aforementioned disease is a CD19-negative cancer, for example, a CD19-negative recurrent cancer. A composition for use or method according to any one of claims 1 to 29.

31. A drug that increases the efficacy of cells expressing the CAR molecule, or A drug that improves one or more side effects associated with the administration of cells expressing the CAR molecule, or Drugs that treat diseases associated with CD19, or Checkpoint inhibitors A composition for use or method according to any one of claims 1 to 30, further comprising administering the following:

32. A composition for use or method according to any one of claims 1 to 31, wherein the subject receives pretreatment, concomitant treatment, or posttreatment with a drug, such as a checkpoint inhibitor, before initiating CART treatment.

33. A composition for use or method according to any one of claims 1 to 32, wherein the patient has CD19-negative cancer cells and cancer cells that are positive for one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

34. A composition for use or method according to any one of claims 1 to 33, further comprising administering to the patient one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, which are positive for the cancer cells.

35. A composition for use or method according to any one of claims 1 to 34, further comprising the step of determining whether the patient has CD19-negative cancer cells, or whether the patient has cancer cells that are positive for one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

36. Separately or mixed, (i) A CAR molecule that binds to CD19, for example, a CAR molecule that binds to CD19 as described herein, for example, one or more cells that express CD19 CAR, and (ii) One or more B cell inhibitors selected from one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1 A composition containing the following:

37. (i) A CAR molecule that binds to CD19, for example, a CAR molecule that binds to CD19 as described herein, for example, a first nucleic acid that encodes a CD19 CAR, and (ii) A second nucleic acid encoding a CAR molecule that binds to one or more B cell antigens selected from CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. A composition comprising the first nucleic acid and the second nucleic acid, wherein the first nucleic acid and the second nucleic acid are in the same or separate nucleic acid molecules.

38. One or more immune effectors, and (i) A CAR molecule that binds to CD19, for example, a CAR molecule that binds to CD19 as described herein, for example, a first nucleic acid encoding a CD19 CAR, or a first polypeptide comprising such a CAR molecule, and (ii) A second nucleic acid encoding a CAR molecule that binds to one or more B cell antigens selected from CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, or a second polypeptide containing such a CAR molecule. A composition containing the following:

39. The first nucleic acid or first polypeptide and the second nucleic acid or second polypeptide are each contained within a first immune effector cell and expressed, for example, by the first immune effector cell; The composition comprises, for example, a first immunoeffector cell expressing the first nucleic acid or first polypeptide, and for example, a second immunoeffector cell expressing the second nucleic acid or second polypeptide; Alternatively, the composition may include both the first nucleic acid or first polypeptide and the second nucleic acid or second polypeptide, for example, without expressing cells. The composition according to claim 38.

40. Cells comprising the nucleic acid composition according to claim 37, which may be human T cells, CD8+ T cells, or NK cells.

41. The cell according to claim 40, further expressing an inhibitor molecule comprising a first polypeptide that includes at least a portion of the inhibitor molecule, associated with a second polypeptide that includes a positive signal from an intracellular signaling domain, wherein the inhibitor molecule may comprise a first polypeptide that includes at least a portion of PD1 and a second polypeptide that includes a co-stimulatory domain and a primary signaling domain.

42. A method for creating cells, comprising introducing the nucleic acid composition described in claim 37 into T cells or NK cells, for example, transduction into T cells or NK cells with the vector described in any one of claims 94 to 100, Assaying for a genetic signature indicating whether a subject treated with the aforementioned cells is likely to relapse or has relapsed; Assaying the gene signature of the cells before injection into the target; or T cells of a cell population including transduced cells ＲＥＧ Reducing the signature It may include the above T ＲＥＧ A method which may include reducing the signature to cause CD25 depletion in the cell population.

43. A composition for use or method according to any one of claims 1 to 42, wherein the CD19 inhibitor comprises a CD19 CAR, the B cell inhibitor comprises a CD123 CAR, and the CD19 CAR or CD123 CAR may include a segmented intracellular signaling domain such that when both the CD19 CAR and the CD123 CAR bind to target cells, e.g., target CD19+CD123+ cells (e.g., B-ALL blast cells), complete activation of cells, e.g., an immunoeffector cell population occurs compared to the activation when the CD19 CAR and the CD123 CAR bind to target cells expressing either CD19 or CD123 (e.g., hematopoietic stem cells).

44. A composition for use or method according to any one of claims 1 to 43, further comprising transplanting cells, such as hematopoietic stem cells or bone marrow cells, into a mammal.

45. A method for treating a subject having DLBCL, for example, primary DLBCL, comprising administering to the subject an effective number of cells expressing a CD19-binding CAR molecule, for example, CD19 CAR, in combination with a PD1 inhibitor, wherein the subject may have, or be identified as having, at least 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% CD3+ / PD1+ DLBCL cells.

46. A method for treating a subject having DLBCL, for example, primary DLBCL, comprising administering to the subject an effective number of one or more cells expressing a CD19-binding CAR molecule, for example, CD19 CAR, wherein the subject may have, or be identified as having, at least 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% CD3+ / PD1+ DLBCL cells.

47. A method for treating a subject having DLBCL, comprising administering to the subject an effective number of cells expressing a CD19-binding CAR molecule, such as CD19 CAR, in combination with a PD-L1 inhibitor, wherein the subject may have, or may be identified as having, cells that are double-positive for CD19 and PD-L1, comprising at least 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% in a tumor, such as the tumor microenvironment.

48. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment comprising a CD20-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the CD20-binding domain comprises one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD20 light chain-binding domain amino acid sequence listed in Tables 13, 15A, or 15B, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD20 heavy chain-binding domain amino acid sequence listed in Tables 12A, 12B, 14A, or 14B.

49. An isolated CAR molecule comprising a CD20-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the CD20-binding domain comprises one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD20-binding domain listed in Table 13, 15A, or 15B, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD20-binding domain listed in Table 12A, 12B, 14A, or 14B.

50. A method for treating a mammal having a disease related to CD20 expression, comprising administering to the mammal an effective amount of cells containing the CAR molecule described in claim 49.

51. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment comprising a CD22-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the CD22-binding domain comprises one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD22 light chain-binding domain amino acid sequence listed in Tables 8A, 8B, 10A, or 10B, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD22 heavy chain-binding domain amino acid sequence listed in Tables 7A, 7B, 7C, 9A, or 9B.

52. An isolated CAR molecule comprising a CD22-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the CD22-binding domain comprises one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of any CD22-binding domain listed in Table 8A, 8B, 10A, or 10B, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of any CD22-binding domain listed in Table 7A, 7B, 7C, 9A, or 9B.

53. A method for treating a mammal having a disease related to CD22 expression, comprising administering to the mammal an effective amount of cells containing the CAR molecule described in claim 52.

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