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

Abstract

The present invention provides compositions and methods for treating diseases associated with CD19 expression, for example by administering a recombinant T cell comprising a CD19CAR described herein in combination with one or more B cell inhibitors, such as an inhibitor of one or more of CCD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. The present disclosure additionally describes novel antigen binding domains and CAR molecules directed against CD20 and CD22, and uses, for example, as monotherapy or combination therapy. The present invention also provides kits and compositions described herein.

Description

[0001] This application is a divisional application of Chinese patent application 202210208342.7, whose application date is April 8, 2016 and whose invention name is “CD20 therapy, CD22 therapy and combined therapy with CD19 chimeric antigen receptor (CAR) expressing cells”.

[0002] This application claims priority to U.S. Serial No. 62 / 144,615 filed on April 8, 2015, U.S. Serial No. 62 / 144,497 filed on April 8, 2015, U.S. Serial No. 62 / 144,639 filed on April 8, 2015, U.S. Serial No. 62 / 207,255 filed on August 19, 2015, and U.S. Serial No. 62 / 263,423 filed on December 4, 2015, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention generally relates to the use of T cells engineered to express a chimeric antigen receptor (CAR), optionally in combination with one or more B cell inhibitors, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a, to treat diseases associated with expression of cluster of differentiation 19 protein (CD19). Background Art

[0004] Many patients with B-cell malignancies are incurable with standard treatments. Furthermore, conventional treatment options often have severe side effects. Attempts have been made at cancer immunotherapy, but several obstacles make achieving clinical effectiveness a very difficult goal. While hundreds of so-called tumor antigens have been identified, they are generally derived from the body's own cells and therefore have poor immunogenicity. Furthermore, tumors employ several mechanisms to render themselves hostile to the initiation and spread of immune attack.

[0005] The latest development of autologous T cell (CART) therapy modified using chimeric antigen receptor (CAR) has shown promising results in treating B cell malignancies and other cancers using the power of the immune system, relying on redirecting T cells to appropriate cell surface molecules on cancer cells such as B cell malignancies (see, e.g., Sadelain et al., CANCER DISCOVERY 3:388-398 (2013)). Clinical results of mouse CART19 (i.e., "CTL019") have shown the hope of establishing complete remission in patients with CLL and childhood 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 chimeric antigen receptor recognition and destruction of target cells on genetically modified T cells, successful therapeutic T cell therapy needs to have the ability to proliferate and continue over time, in order to investigate leukemia relapse. Due to anergy, the quality of the changes in T cells caused by suppression or exhaustion will have an impact on the performance of the T cells converted by CAR, and skilled doctors have limited control over the performance at this time. In order to be effective, the patient T cells converted by CAR need to be persistent and maintain the ability to proliferate in response to cognate antigens. It has been shown that ALL patient T cells can be carried out with CART19 comprising mouse scFv (see, for example, Grupp et al., NEJM 368: 1509-1518 (2013)). SUMMARY OF THE INVENTION

[0007] The disclosure relates, at least in part, to methods for treating disorders associated with expression of cluster of differentiation antigens 19 protein (CD19) (e.g., OMIM Acc.No.107265, Swiss Prot.Acc No.P15391). In certain embodiments, the disorder is cancer, such as a blood cancer. In some embodiments, the method includes administering a chimeric antigen receptor (CAR) molecule for binding CD19 in combination with a B cell inhibitor, such as one or more (e.g., one, two, three or more) B cell inhibitors. In some embodiments, the B cell inhibitor is selected from an inhibitor of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3 or ROR1, or a combination thereof. In some embodiments, the combination maintains or has better clinical effectiveness than any single therapy. In some embodiments, the methods herein relate to the use of engineered cells, such as T cells expressing a CAR molecule that binds CD19 and a B cell inhibitor (e.g., an antibody (e.g., a monospecific or bispecific antibody) or a CAR-expressing cell that binds a second B target, such as a CAR-expressing immune effector cell, a combination thereof) to treat a condition associated with the expression of CD19. The present disclosure further describes novel antigen binding domains and CAR molecules for CD20 and CD22, and, for example, for use as a monotherapy or combination therapy.

[0008] Therefore, on the one hand, the present invention relates to a method for treating a subject (eg, a mammal) with a disease associated with CD19 expression. The method includes administering to the subject a CD19 inhibitor in combination with a B cell inhibitor, for example, with reference to the CAR molecules of CD19 described herein. For example, the method includes administering to the subject one or more CAR molecules of an effective number of expressions in combination with a B cell inhibitor, for example, with reference to the CAR molecules of CD19 described herein (eg, wild-type or mutant CD19). In certain embodiments, the B cell inhibitor is selected from a CD10 inhibitor, e.g., one or more CD10 inhibitors described herein; a CD20 inhibitor, e.g., one or more CD20 inhibitors described herein; a CD22 inhibitor, e.g., one or more CD22 inhibitors described herein; a CD34 inhibitor, e.g., one or more CD34 inhibitors described herein; a CD123 inhibitor, e.g., one or more CD123 inhibitors described herein; a FLT-3 inhibitor, e.g., one or more FLT-3 inhibitors described herein; a ROR1 inhibitor, e.g., one or more ROR1 inhibitors described herein; a CD79b inhibitor, e.g., one or more CD79b inhibitors described herein; a CD179b inhibitor, e.g., one or more CD179b inhibitors described herein; a CD79a inhibitor, e.g., one or more CD79a inhibitors described herein, or any combination thereof. In certain aspects, disclosed is a method of treating a subject having a B-cell leukemia or B-cell lymphoma, comprising administering to the subject an effective number of cells expressing one or more CAR molecules that bind CD19, in combination with one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a.

[0009] In related aspects, the present disclosure provides methods for reducing CD19 expressing cell proliferation, for example, by applying a combination therapy as described herein to a subject (e.g., a patient in need thereof), such as a CD19 inhibitor and a B cell inhibitor, for example, one or more B cell inhibitor combinations as described herein. On the other hand, the present disclosure provides methods for selectively killing cells expressing CD19, for example, by applying a combination therapy as described herein to a subject (e.g., a patient in need thereof), such as a CD19 inhibitor and a B cell inhibitor, for example, one or more B cell inhibitor combinations as described herein. In certain aspects, the present disclosure provides methods for providing anti-tumor immunity in a subject, such as a mammal, including administering an effective amount of a combination as described herein (e.g., one or more CAR expressing cells) to a mammal.

[0010] In one aspect, the present disclosure provides a method of preventing CD19-negative relapse in a mammal, comprising administering to the mammal one or more B cell inhibitors, wherein the B cell inhibitor comprises an inhibitor of one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0011] On the other hand, the present disclosure provides a method for treating a subject having a disease associated with CD19 expression, such as DLBCL (e.g., primary DLBCL). The method includes administering to the subject an effective amount of one or more CAR molecules expressing binding CD19, such as cells of CD19 CAR, optionally in combination with a PD1 inhibitor. Optionally, the subject has or is identified as having at least 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of cancer cells, such as DLBCL cells, which are CD3+ / PD1+.

[0012] On the one hand, the present disclosure provides a method for treating a subject with a disease (e.g., DLBCL) associated with CD19 expression. The method includes administering to the subject an effective number of one or more cells that express a CAR molecule that binds CD19, such as CD19 CAR, in combination with a PD-L1 inhibitor. Optionally, the subject has or is identified as having less than 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% cancer cells, such as a cancer microenvironment, which is double positive for CD19 and PD-L1.

[0013] In one aspect, the present disclosure provides one or more B cell inhibitors, wherein the B cell inhibitor comprises one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a, for treating a subject having a disease associated with CD19 expression, and wherein the subject has received or is receiving or is about to receive a cell expressing a CAR molecule that binds to CD19, e.g., a CD19 CAR.

[0014] Timing and dosage of combined administration

[0015] One or more therapies described herein can be administered to a subject substantially simultaneously or in any order. For example, a CD19 inhibitor, such as a CD19 CAR expressing cell described herein, one or more B cell inhibitors and / or optionally at least one additional therapeutic agent can be administered simultaneously in the same or separate compositions, or sequentially.

[0016] For sequential administration, CAR-expressing cells as described herein (e.g., CD19 CAR-expressing cells, CD20 CAR-expressing cells, or CD22 CAR-expressing cells) may be administered first, and additional agents may be administered secondarily, or the order of administration may be reversed. In some embodiments, when the second therapy is introduced, the first therapy (e.g., CAR-expressing cells, such as CD19 CART cells, CD20 CART cells, or CD22 CART cells) is continued, and in other embodiments, the first therapy is withdrawn before or after the second therapy is introduced or withdrawn simultaneously with the second therapy. In the case of sequential administration, in some embodiments, the second therapy is started after a predetermined amount of time or after the subject shows one or more indications that recurrence has occurred or may occur. Indications may be, for example, the presence of cancer cells with interference in the target of the first therapy, such as CD19, CD20, or CD22. Interference may be, for example, frameshift mutations and / or premature termination codons.

[0017] In other embodiments, two or more therapies (such as CD19 CAR expressing cells and B cell inhibitors) are administered simultaneously. Without being bound by theory, in some embodiments, the simultaneous administration of therapy can reduce the likelihood of recurrence and / or delay recurrence.

[0018] When administered in combination, the first therapy (e.g., CAR therapy, for example, for CD19, CD20 or CD22 CAR-expressing cells) and another agent (e.g., a second or third agent, such as a B cell inhibitor), or all can be administered in an amount higher, lower or the same amount or dosage as each agent used alone (e.g., as a monotherapy). In certain embodiments, the first therapy, the second therapy, the optional third therapy or all of the administered amount or dosage ratio is 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 alone as a monotherapy. In other embodiments, the first therapy, the second therapy, the optional third therapy or all of the administered amount or dosage ratio results in a desired effect (e.g., treatment of cancer) that is required to achieve the same therapeutic effect, for example, the amount or dosage of each agent used alone as a monotherapy is lower (e.g., at least 20%, at least 30%, at least 40% or at least 50%). In certain embodiments, the side effects seen when administering conventional (monotherapy) doses are compared to the side effects seen when administering conventional (monotherapy) doses.

[0019] In one embodiment, the therapy comprises a population of cells. In embodiments, the cells are immune effector cells, e.g., CAR-expressing cells.

[0020] Alternatively, or in combination with the methods described herein, methods are disclosed that include a diagnostic step or a patient selection step, for example as described below.

[0021] In one aspect, the invention provides a method for assessing the relapser status (e.g., relapser or non-relapser after CAR therapy) of a subject (e.g., patient). In one embodiment, after treatment with CAR therapy (e.g., CD19 CART therapy, such as described herein, such as CTL019 therapy), the method identifies a subject who has relapsed ("relapser") or may relapse or not relapse ("non-relapser") or may not relapse, such as a patient. In one embodiment, relapser status (e.g., relapser or non-relapser after CART therapy) is determined by measuring one or more features of CD19.

[0022] In one embodiment, one or more features of CD19 include changes in nucleic acid sequence (e.g., mutations, such as insertions, deletions or substitutions, or a combination thereof), changes in nucleic acid levels, changes in protein sequence, or changes in protein levels, or a combination thereof. In one embodiment, the relapser has one or more mutations in CD19, such as one or more mutations (e.g., insertions or deletions) in exon 2 of CD19. In one embodiment, the relapser has one or more mutations in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of CD19. In one embodiment, the mutation produces a premature stop codon, such as a frameshift caused by insertion or deletion, such as a frameshift in exon 2 of CD19. In one embodiment, the mutation is a mutation of Table 31.

[0023] In one embodiment, the feature of CD19 is compared with a reference feature. For example, when the feature is a sequence (e.g., a protein or nucleic acid sequence from a biological sample), the reference feature can be the wild-type sequence of CD19 (e.g., a protein or nucleic acid sequence). The feature can be the percentage of cells with a mutant sequence in the sample. When the feature is a level (e.g., a protein or nucleic acid level), the reference feature can be the wild-type level of CD19 (e.g., a protein or nucleic acid level). The feature can be the level of a protein or nucleic acid in the sample. The feature can be the percentage of cells with a protein or nucleic acid level higher than a given threshold in the sample.

[0024] In one embodiment, there is provided a method for identifying a subject with cancer (e.g., a hematological cancer such as CLL or ALL) as a relapser or non-relapser after treatment including CAR therapy, such as CD19 CART therapy. The method includes: (1) obtaining a sample from the subject (e.g., a single apheresis sample obtained from the subject's blood; and / or such as a finished product sample, such as a genetically engineered T cell obtained from the subject's blood); (2) determining the characteristics of CD19, such as a sequence or level as described herein; and (3) (optionally) comparing the characteristics of the determined CD19 with a reference characteristic; wherein the difference between the determined characteristic and the reference characteristic, such as a statistically significant difference, indicates relapse to CAR therapy; and (4) for example, based on the determined CD19 characteristics, the subject is identified as a relapser or non-relapser of CAR therapy. In one embodiment, the presence or absence of the feature of CD19 is the presence or absence of a premature stop codon, such as by causing an insertion or deletion of a frameshift. In one embodiment, the presence of the feature of CD19 is a mutation of Table 31.

[0025] In one embodiment, the method provided includes (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, such as a genetically engineered T cell obtained from the subject's blood, such as a manufactured CART19 product); (2) determining a feature of CD19, such as a sequence or level as described herein; and (3) (optionally) comparing the determined CD19 characteristic with a reference feature; wherein the presence of a feature of CD19 (e.g., a difference between the determined features compared to a reference feature, such as a statistically significant difference) is indicative of recurrence of CAR therapy. In one embodiment, the presence of a feature of CD19 is the presence of a premature stop codon, such as by an insertion or deletion that results in a frameshift. In one embodiment, the presence of a feature of CD19 is a mutation of Table 31.

[0026] In one embodiment, there is provided a method for determining the recurrence of a subject with cancer (e.g., hematological cancers such as CLL or ALL) after treatment including CAR therapy (e.g., CD19 CAR therapy as described herein). The method includes determining the features of CD19 in the sample obtained before recurrence. In one embodiment, the presence of the features of CD19 (e.g., the difference between the features determined compared to a reference feature, such as a statistically significant difference) indicates recurrence after CAR therapy. In one embodiment, the presence of the features of CD19 is the presence of premature stop codons, such as by causing the insertion or deletion of a frameshift. In one embodiment, the presence of the features of CD19 is the mutation of Table 31.

[0027] In one embodiment, a method for assessing a subject suffering from cancer, such as a hematological cancer such as CLL or ALL is provided. The method includes obtaining a relapser status value for the subject, including a measured value of one or features of CD19 (such as one or more features of CD19 as described herein), thereby assessing the subject.

[0028] In one embodiment, a method for evaluating or monitoring the effectiveness of a CAR therapy (e.g., a CD19 CART therapy) in a subject having cancer is provided, the method comprising obtaining a relapser status value for the subject, comprising measuring one or more characteristics of CD19, e.g., one or more characteristics of CD19 described herein, thereby evaluating or monitoring the effectiveness of the CAR therapy in the subject.

[0029] In one embodiment, a method for providing a prediction of the success rate of a CAR therapy (e.g., a CD19 CART therapy described herein) in a subject having cancer is provided, the method comprising the steps of providing a biological sample from the subject; determining one or more characteristics of CD19, e.g., one or more characteristics of CD19 described herein; and providing a prognosis for the subject based on the determined characteristics.

[0030] In some aspects, the present disclosure provides methods or assays, e.g., for identifying a subject having cancer who has an increased or decreased likelihood of responding to a treatment comprising a chimeric antigen receptor (CAR) therapy, comprising:

[0031] (1) Obtaining a sample from a subject;

[0032] (2) Determine the value of one or more of the following:

[0033] (i) the level of one or more markers listed in Table 29 in the sample;

[0034] (ii) a characteristic of CD19, such as a mutation, for example, one that results in a frameshift or premature stop codon or both, or

[0035] (iii)T REG the level or activity of cells; and

[0036] (3) (optionally) comparing the determined value, e.g., the level, activity, or property of (i), (ii), or (iii), or a combination thereof, to a reference value, wherein a difference, e.g., a statistically significant difference, between the determined value and the reference value predicts responsiveness of the subject to the CAR therapy; and

[0037] (4) Identifying the subject as a complete responder, partial responder or non-responder to CAR therapy, or a relapser or non-relapser based on the determined value.

[0038] In certain embodiments, any of the above methods may further comprise the following:

[0039] (i) If (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 premature stop codon or both, or (iii) TREG If no difference is detected in one, two or more (all) values ​​of the level of CD19-expressing cells (e.g., no statistically significant difference), then administering to the subject a therapeutically effective dose of a CAR therapy, e.g., a therapy comprising CD19-expressing cells;

[0040] (ii) if (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 premature stop codon or both, or (iii) TREG If a difference (e.g., a statistically significant difference) is detected in one, two or more (all) values ​​of the levels of cells expressing CD19, then a therapeutically effective dose of a CAR therapy is administered to the subject, e.g., comprising cells expressing CD19 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

[0041] (iii) if (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 premature stop codon or both, or (iii) TREG If a difference (e.g., a statistically significant difference) is detected in one, two or more (all) of the values ​​of the levels of CD19-expressing cells, then the first therapy, e.g., a therapy comprising CD19-expressing cells, is discontinued and 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) is administered.

[0042] Administration steps (i)-(iii) may be performed before or after the patient evaluation step, as described in the exemplary embodiments below.

[0043] In certain aspects, a method of treating a subject suffering from cancer is disclosed. The method comprises:

[0044] (a) obtaining, e.g., determining, whether the subject has one, two, or more (all) of the following values:

[0045] (i) the level of one or more markers listed in Table 29;

[0046] (ii) characteristics of CD19, such as mutations that cause frameshifts or premature stop codons, or both, or

[0047] (iii) T in biological samples REG the level or activity of cells, and

[0048] (b) in response to the value, further comprising the following:

[0049] (i) If (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 premature stop codon or both, or (iii) TREG If no difference is detected in one, two or more (all) values ​​of the level of CD19-expressing cells (e.g., no statistically significant difference), then administering to the subject a therapeutically effective dose of a CAR therapy, e.g., a therapy comprising CD19-expressing cells;

[0050] (ii) if (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 premature stop codon or both, or (iii) TREG If a difference (e.g., a statistically significant difference) is detected in one, two or more (all) values ​​of the levels of cells expressing CD19, then a therapeutically effective dose of a CAR therapy is administered to the subject, e.g., a therapy comprising cells expressing CD19 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

[0051] (iii) if (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 premature stop codon or both, or (iii) TREG If a difference (e.g., a statistically significant difference) is detected in one, two or more (all) of the values ​​of the levels of CD19-expressing cells, then the first therapy, e.g., a therapy comprising CD19-expressing cells, is discontinued and 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) is administered.

[0052] In another aspect, a method for treating a subject suffering from cancer is provided. The method comprises:

[0053] (a) administering to the subject a therapeutically effective dose of a CAR therapy, e.g., a therapy comprising CD19-expressing cells,

[0054] (b) obtaining (e.g., determining whether a subject has) one, two, or more (all) of the following values:

[0055] (i) the level of one or more markers listed in Table 29;

[0056] (ii) a characteristic of CD19, such as a mutation, e.g., a mutation causing a frameshift or premature stop codon or both, or

[0057] (iii) T in biological samples REG the level or activity of cells, and

[0058] (c) in response to the value or determination in step (b)(I-III), performing one or more of the following:

[0059] (i) if (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 causing a frameshift or premature stop codon or both, or (III) TREG If no difference is detected in one or more of the levels of CAR T cells (e.g., no statistically significant difference), administering to the subject a therapeutically effective dose of a CAR therapy, e.g., a therapy comprising CD19-expressing cells;

[0060] (ii) if (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 premature stop codon or both, or (III) TREG If a difference (e.g., a statistically significant difference) is detected in one or more of the levels of B cell expressed in the subject, a therapeutically effective dose of a CAR therapy is administered to the subject, e.g., a therapy comprising cells expressing CD19 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

[0061] (iii) if (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 premature stop codon or both, or (III) T in the biological sample REGIf a difference (e.g., a statistically significant difference) is detected in the levels of one or more of the B cell lines expressed in the cells, the first therapy, e.g., a therapy comprising CD19-expressing cells, is discontinued and 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) is administered.

[0062] In some embodiments of any of the above methods, the sample is a biological sample selected from blood, plasma, or serum samples. In a specific embodiment, the biological sample is a blood sample. In one embodiment, the sample is an apheresis sample, such as a T cell obtained from the subject's blood. In one embodiment, the sample is a finished product sample, such as a genetically engineered T cell obtained from the subject's blood, such as a manufactured CAR product, such as a manufactured CART19 product.

[0063] In one embodiment, the method herein can be used to determine whether a patient is likely to respond to CAR therapy (e.g., CD19CART), for example, whether a patient who has not yet received CAR therapy is likely to respond to CAR therapy, or whether a patient who has received CAR therapy is likely to respond to sustained CAR therapy. In general, the same CD19 features that predict recurrence predict that the patient is unlikely to respond to CD19 CAR therapy. Patients identified as being unlikely to respond to CD19 CAR therapy can be administered different types of therapies, such as B cell inhibitors (e.g., CD10 as described herein, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, one or more inhibitors of CD179b or CD79a).

[0064] On the other hand, there is provided a method for treating a subject having cancer, such as a blood cancer. In one embodiment, the method includes determining whether the subject has a difference in the feature of CD19 relative to a reference feature, such as a statistically significant difference, and if there is a difference between the determined feature and the reference feature, such as a statistically significant difference, then administering a therapeutically effective dose of CAR therapy, such as CART, to the subject to treat the subject. In one embodiment, the feature is a CD19 sequence, such as a protein or nucleic acid sequence. In one embodiment, the method includes determining whether there is a frameshifted CD19, for example, a CD19 comprising a premature stop codon.

[0065] In the embodiment of any of the above methods, treatment includes administering CD19 CAR expressing cells, optionally in combination with one or more B cell inhibitors. In one embodiment, CD19 CAR therapy is administered simultaneously with one or more B cell inhibitors (e.g., CD10 as described herein, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or one or more inhibitors of CD79a). In one embodiment, CD19 CAR therapy is administered before one or more B cell inhibitors. In one embodiment, CD19 CAR therapy is administered after one or more B cell inhibitors.

[0066] In embodiments where there is a discrepancy between the determined profile and the reference profile, the method comprises modifying the CAR product prior to infusion into the subject. In one embodiment where there is a discrepancy between the determined profile and the reference profile, the method comprises modifying the manufacture of the CAR product prior to infusion into the subject. In one embodiment, if there is a discrepancy between the determined profile and the reference profile, the method comprises adjusting the CAR infusion dose to achieve an anti-cancer effect.

[0067] In one embodiment, the method of treatment comprises determining whether the subject has an increased likelihood of responding to a CAR therapy, such as a CD19 CART therapy, such as a CD19 CART therapy described herein, by comparing a signature of CD19 in a sample from the subject relative to a reference signature, wherein a difference in the signature relative to the reference signature indicates an increased likelihood of response; and administering a therapeutically effective dose of the CAR therapy to the subject, thereby treating the subject.

[0068] In one embodiment, the method of treatment comprises obtaining a sample from a subject; determining a characteristic of CD19 (e.g., the presence or absence of a frameshift or premature stop codon) relative to a reference characteristic; and administering a therapeutically effective dose of CAR-expressing cells if the subject is identified as having a statistically significant difference between the CD19 characteristic of the sample and the reference characteristic in the sample.

[0069] CD19 features can be used to design treatments for patients. For example, in one embodiment, when a patient sample includes wild-type CD19, a CD19 inhibitor is administered to the patient, such as a cell expressing CD19 CAR, such as CD19 CART. In one embodiment, when a patient sample includes mutant CD19 (e.g., frameshift CD19, such as CD19 comprising premature stop codons), a therapy other than a CD19 inhibitor is administered to the patient, for example, the patient is administered another B cell inhibitor. In one embodiment, when a patient sample includes at least normal levels of CD19, a CD19 inhibitor is administered to the patient, such as a cell expressing CD19 CAR, such as CD19 CART. In one embodiment, when a patient sample includes less than normal levels of CD19, a therapy other than a CD19 inhibitor is administered to the patient, for example, the patient is administered another B cell inhibitor (e.g., CD10 as described herein, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or one or more inhibitors of CD79a).

[0070] In one embodiment, the method of treatment comprises obtaining a value for the subject's relapser status, including a measure of a CD19 signature, and in response to the determination of relapser status, performing one, two, three, or more of the following: (1) identifying the subject as a relapser or non-relapser; (2) administering a CAR therapy; (3) selecting or changing the dose of the CAR therapy; (4) selecting or changing the schedule or time course of the CAR therapy; (5) administering an additional agent in combination with the CAR therapy, e.g., administering one or more B cell inhibitors, or checkpoint inhibitors, e.g., checkpoint inhibitors described herein, or kinase inhibitors, e.g., kinase inhibitors described herein, to a relapser; (6) administering a therapy to a relapser that increases the number of naive T cells in the subject prior to treatment with the CAR therapy; modifying the method of manufacturing the CAR therapy, e.g., for a subject identified as a relapser, e.g., enriching for naive T cells prior to introducing a nucleic acid encoding the CAR; or (7) selecting an alternative therapy, e.g., a standard of care for a particular cancer (e.g., as described herein) (e.g., for a relapser); thereby treating the subject's cancer.

[0071] In some embodiments, the method includes administering one, two, three or more B cell inhibitors (e.g., CD10 as described herein, CD20, CD22, CD34, CD123, one or more inhibitors of FLT-3 or ROR1). For example, in one embodiment, the method includes administering a CD19 inhibitor, such as a cell expressing CD19 CAR in combination with a CD10 inhibitor or a CD10 inhibitor and CD20 as described herein, CD22, CD34, CD123, any combination of an inhibitor of FLT-3 or ROR1. In another embodiment, the method includes administering a CD19 inhibitor, such as a cell expressing CD19 CAR in combination with a CD20 inhibitor or a CD20 inhibitor and any combination of CD10, CD22, CD34, CD123, FLT-3 or ROR1 inhibitor as described herein. In another embodiment, the method includes administering a CD19 inhibitor, such as a cell expressing CD19 CAR in combination with a CD22 inhibitor, or a CD22 inhibitor and any combination of CD10, CD22, CD34, CD123, FLT-3 or ROR1 inhibitors as described herein. In another embodiment, the method includes administering a CD19 inhibitor, such as a cell expressing CD19 CAR in combination with a CD34 inhibitor or a CD34 inhibitor and any combination of CD10, CD22, CD34, CD123, FLT-3 or ROR1 inhibitors as described herein. In another embodiment, the method includes administering a CD19 inhibitor, such as a cell expressing CD19 CAR in combination with a CD123 inhibitor, or a CD123 inhibitor and any combination of CD10, CD22, CD34, CD123, FLT-3 or ROR1 inhibitors as described herein. In another embodiment, the method includes administering a CD19 inhibitor, for example, a combination of cells expressing CD19CAR and a FLT-3 inhibitor or a FLT-3 inhibitor and CD10 as described herein, CD22, CD34, CD123, or any combination of ROR1 inhibitors. In another embodiment, the method includes administering a CD19 inhibitor, for example, a cell expressing CD19 CAR, in combination with a ROR1 inhibitor or a ROR1 inhibitor and CD10 as described herein, CD22, CD34, CD123, any combination of FLT-3 inhibitors. 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).

[0072] In some embodiments, the methods of treatment described herein further include one or both of the following: determining the level of immune checkpoint molecules (e.g., PD-L1, PD1, LAG3, or TIM3) in a patient sample; and administering immune checkpoint inhibitors (e.g., inhibitors of one or more of PD-L1, PD1, LAG3, and TIM3) to the patient. For example, the method may include treating a patient with one or more CAR-expressing cells as described herein (e.g., CD19 CAR in combination with B cell inhibitors, CD20 CAR, or CD22 CAR) and determining the level of immune checkpoint molecules in the patient before and after treatment. In some embodiments, the method includes administering an immune checkpoint inhibitor to a patient having an elevated immune checkpoint molecule level compared to a reference level, for example, administering a PD-L1 inhibitor in response to elevated PD-L1 levels, administering a PD1 inhibitor in response to elevated PD1 levels, administering a LAG3 inhibitor in response to elevated LAG3 levels, or administering a TIM3 inhibitor in response to elevated TIM3 levels. In some embodiments, the method comprises administering an immune checkpoint inhibitor (e.g., a CD19 CAR in combination with a B cell inhibitor, a CD20 CAR, or a CD22 CAR) to a patient who has received, is receiving, or will receive treatment with one or more CAR-expressing cells described herein, wherein the patient has or is identified as having an elevated level of an immune checkpoint molecule compared to a reference level.

[0073] Composition

[0074] In some aspects, the present disclosure provides, for example, a composition comprising: (i) expressing a CAR molecule that binds CD19, for example, a CAR molecule that binds CD19 as described herein, for example, one or more cells of CD19 CAR, and (ii) a B cell inhibitor, for example, one or more inhibitors of CD10, CD20, CD22, CD34, CD123, FLT-3 or ROR1. In an embodiment, (i) and (ii) are provided separately, and (i) and (ii) are mixed in an embodiment.

[0075] In some aspects, the present disclosure provides, for example, nucleic acids encoding the following: (i) CAR molecules in combination with CD19, for example, CAR molecules in combination with CD19 as described herein, such as CD19 CAR, and (ii) one or more B cell inhibitors, such as CD10, CD20, CD22, CD34, CD123, FLT-3 or ROR1. In some aspects, the present disclosure provides, for example, nucleic acids encoding the following: (i) CAR molecules in combination with CD19, for example, CAR molecules in combination with CD19 as described herein, such as CD19 CAR, and (ii) CAR molecules that bind to B cell antigens, such as CD10, CD20, CD22, CD34, CD123, FLT-3 or ROR1. In embodiments, nucleic acid comprises RNA or DNA.

[0076] In some aspects, the present disclosure provides, for example, nucleic acids encoding the following: (i) CAR molecules in combination with CD19, for example, CAR molecules in combination with CD19 as described herein, such as CD19 CAR, and (ii) CAR molecules, which bind B cell antigens, such as CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, one or more of CD179b or CD79a. In embodiments, nucleic acid comprises RNA or DNA. In embodiments, the nucleic acid sequences encoding (i) and (ii) are located in the same orientation, such as the transcription of the nucleic acid sequences encoding (i) and (ii) is carried out in the same direction. In embodiments, the nucleic acid sequences encoding (i) and (ii) are located in different orientations. In embodiments, a single promoter controls the expression of the nucleic acid sequences encoding (i) and (ii). In embodiments, nucleic acids encoding protease cleavage sites (such as T2A, P2A, E2A or F2A cleavage sites) are located between the nucleic acid sequences encoding (i) and (ii). In embodiments, the protease cleavage site is placed so that the cell can express a fusion protein comprising (i) and (ii), which is subsequently processed into two peptides by proteolytic 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 located upstream of the nucleic acid sequence encoding (i). In embodiments, the first promoter controls the expression of the nucleic acid sequence encoding (i), and the second promoter controls the expression of the nucleic acid sequence encoding (ii). In embodiments, nucleic acid is a plasmid. In embodiments, nucleic acid comprises viral packaging elements. In some aspects, the present disclosure provides cells comprising nucleic acids as described herein, such as cells comprising nucleic acids of (i) and (ii) as described above, such as immune effector cells. The cell can comprise a protease (e.g., endogenous or exogenous) that cuts T2A, P2A, E2A or F2A cleavage sites.

[0077] In some aspects, the present disclosure provides, for example, a composition comprising: (i) encoding a CAR molecule that binds CD19, for example, a CAR molecule that binds CD19 as described herein, for example, a first nucleic acid of CD19 CAR, and (ii) encoding one or more B cell inhibitors, for example, a second nucleic acid of an inhibitor of one or more of CD10, CD20, CD22, CD34, CD123, FLT-3 or ROR1. In some aspects, the present disclosure provides, for example, a composition comprising: (i) encoding a CAR molecule that binds CD19, for example, a CAR molecule that binds CD19 as described herein, for example, a first nucleic acid of CD19 CAR, and (ii) binding to B cell antigens, for example, one or more CAR molecules of 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.

[0078] In some aspects, the present disclosure provides, for example, vectors comprising one or more nucleic acids described herein. In certain aspects, the present disclosure also provides cells comprising a vector or nucleic acid as described herein.

[0079] The present disclosure also provides compositions in certain aspects, and it includes one or more immune effector cells, and:(i) first nucleic acid or the first polypeptide encoding the first polypeptide, the first polypeptide including the CAR molecules for binding CD19, for example, with reference to the CAR molecules of CD19 as described herein, such as CD19 CAR, and (ii) the first nucleic acid or the second polypeptide encoding the second polypeptide, the second polypeptide including binding B cell antigens, such as CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a one or more CAR molecules. In embodiments, the first nucleic acid or the first polypeptide and the second nucleic acid or the second polypeptide are each contained in the first immune effector cell, for example, expressed by the first immune effector cell. In embodiments, the composition includes the first immune effector cell containing, for example, expressing the first nucleic acid or the first polypeptide and the second immune effector cell containing, for example, expressing the second nucleic acid or the second polypeptide. In embodiments, the composition does not include cells containing, for example, expressing the first nucleic acid or the first polypeptide and the second nucleic acid or the second polypeptide.

[0080] manufacture

[0081] In some aspects, the present disclosure provides a method for preparing a cell, including transducing immune effector cells, such as T cells or NK cells, with a vector as described herein (e.g., a vector encoding CAR). In some aspects, the present disclosure provides a method for preparing a cell, including introducing nucleic acid as described herein (e.g., nucleic acid encoding CAR) into immune effector cells such as T cells or NK cells. In some aspects, the present disclosure provides a method for producing an RNA engineered cell colony, including introducing in vitro transcribed RNA or synthetic RNA into a cell, wherein RNA includes nucleic acid as described herein, such as nucleic acid encoding CAR.

[0082] In some embodiments, the preparation method disclosed herein further comprises contacting a cell population (e.g., a cell expressing CD19 CAR, a cell expressing CD20 CAR, a cell expressing CD22 CAR, a B cell inhibitor cell, or both a CD19 CAR expressing cell and a B cell inhibitor cell) with a nucleic acid encoding a telomerase subunit, such as hTERT. The nucleic acid encoding the telomerase subunit can be DNA.

[0083] In some embodiments, the preparation methods disclosed herein further comprise culturing the cell population (e.g., cells expressing CD19 CAR, cells expressing CD20 CAR, cells expressing CD22 CAR, B cell suppressor cells, or both cells expressing CD19 CAR and B cell suppressor cells) in serum containing 2% hAB serum.

[0084] Indications

[0085] In one embodiment, the disease associated with CD19 expression is selected from a proliferative disease such as cancer or malignancy or a precancerous condition such as myelodysplasia, myelodysplastic syndrome or preleukemia, or an indication associated with a non-cancer associated with the expression of CD19. In one embodiment, the disease is a solid or liquid tumor. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the disease is a blood cancer. In one embodiment, the blood cancer is a leukemia. In one embodiment, the cancer is selected from 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), acute lymphoblastic leukemia (ALL) (e.g., relapsed and refractory ALL); one or more chronic leukemias, including but not limited to chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Additional hematological cancers or conditions include, but are not limited to, mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia". Preleukemia includes a group of different hematological conditions associated with ineffective production (or dysplasia) of myeloid blood cells. In embodiments, diseases associated with CD19 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases expressing CD19; and any combination thereof.

[0086] In one embodiment, the disease associated with CD19 expression is a lymphoma, such as MCL or Hodgkin's lymphoma. In one embodiment, the disease associated with CD19 expression is a leukemia, such as SLL, CLL and / or ALL.

[0087] In one embodiment, the disease associated with a tumor antigen (e.g., a tumor antigen described herein) is selected from a proliferative disease such as a cancer or malignancy or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia, or is a non-cancer related indication associated with expression of a tumor antigen described herein. In one embodiment, the disease associated with a tumor antigen described herein is a solid tumor, e.g., a solid tumor described herein, such as prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer, or lung cancer.

[0088] In one embodiment, the cancer is selected from AML, ALL, B-ALL, T-ALL, B-cell prolymphocytic leukemia, chronic lymphocytic leukemia, CML, hairy cell leukemia, Hodgkin lymphoma, mast cell disorder, myelodysplastic syndrome, myeloproliferative neoplasms, plasma cell myeloma, plasmacytoid dendritic cell neoplasm, or a combination thereof.

[0089] In one embodiment, the subject (e.g., a subject treated with a CD19 CAR, optionally in combination with a second agent such as a PD1 inhibitor or a PD-L1 inhibitor) has or is identified as having at least 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cancer cells, e.g., DLBCL cells, that are CD3+ / PD1+.

[0090] In one embodiment, the subject has relapsed or been identified as having relapsed after treatment with one or more cells expressing a CAR molecule (e.g., CD19 CAR) binding to CD19. In one embodiment, the subject has relapsed or been identified as having relapsed based on one or more reappearances of blasts in blood, bone marrow (>5%) or any extramedullary site after a complete response. In one embodiment, the subject has relapsed or been identified as having relapsed based on detection of CD19-blasts above a predetermined threshold (e.g., more than 1%, 2%, 3%, 4%, 5% or 10%).

[0091] CAR therapy

[0092] In certain embodiments, the method of treatment includes CAR therapy, such as administering one or more cells expressing one or more CAR molecules. The cells expressing one or more CAR molecules can be immune effector cells, such as T cells or NK cells. In one embodiment, the subject is a human.

[0093] In one embodiment, the cell expressing the CAR molecule includes a vector comprising a nucleic acid sequence encoding the CAR molecule. In one embodiment, the vector is selected from DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector. In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further comprises a promoter. In one embodiment, the promoter is an EF-1 promoter. In one embodiment, the EF-1 promoter comprises a sequence of SEQ ID NO:100. In one embodiment, the vector is an in vitro transcribed vector, such as a vector for RNA that transcribes a nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a poly (A) tail, such as a poly A tail as described herein, for example, comprising about 150 adenosine bases. In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a 3'UTR, such as a 3'UTR as described herein, for example, comprising at least one repeat sequence of the 3'UTR from human β-globulin. In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a promoter. In one embodiment, the nucleic acid sequence comprises a T2A sequence.

[0094] In one embodiment, the cell expressing the CAR molecule is a cell as described herein, such as a human T cell or a human NK cell, such as a human T cell as described herein or a human NK cell as described herein. In one embodiment, human T cells are CD8+T cells. In one embodiment, human T cells are CD4+T cells. In one embodiment, human T cells are CD4+ / CD8+T cells. In one embodiment, human T cells are a mixture of CD8+ and CD4+T cells. In one embodiment, the cell is an autologous T cell. In one embodiment, the cell is an allogeneic T cell. In one embodiment, the cell is a T cell, and the T cell is diacylglycerol kinase (DGK) defective. In one embodiment, the cell is a T cell, and the T cell is Ikaros defective. In one embodiment, the cell is a T cell, and the T cell is DGK and Ikaros defective.

[0095] In another embodiment, a cell expressing a CAR molecule, e.g., as described herein, can further express another agent, e.g., an agent that enhances the activity of the CAR-expressing cell.

[0096] In one embodiment, the method includes administering a cell expressing a CAR molecule as described herein in combination with an agent that enhances the activity of the CAR-expressing cell, 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 CAR-expressing cell, for example, simultaneously or soon after administration. Alternatively, the cytokine can be delivered after an extended period of time, for example, after assessing the subject's response to the CAR-expressing cell.

[0097] For example, in one embodiment, the active agent that enhances the activity of CAR-expressing cells can be an active 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 TGFRβ. In one embodiment, the active agent that inhibits immunosuppressive molecules includes a first polypeptide that is bound to a second polypeptide (e.g., an intracellular signaling domain as described herein) that provides a positive signal to the cell, such as an inhibitory molecule. In one embodiment, the agent comprises a first polypeptide, e.g., of an immunosuppressive molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFRβ, 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 described herein (e.g., including a costimulatory domain (e.g., 41BB, CD27, or CD28, e.g., described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent 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 of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein).

[0098] In one embodiment, lymphocyte infusion (e.g., allogeneic lymphocyte infusion) is used to treat cancer, wherein lymphocyte infusion includes at least one CD19 CAR expressing cell as described herein and optionally at least one cell expressing a CAR for a B cell antigen. In one embodiment, autologous lymphocyte infusion is used to treat cancer, wherein autologous lymphocyte infusion includes at least one cell expressing CD19 and optionally at least one cell expressing a CAR for a B cell antigen.

[0099] In one embodiment, CAR-expressing cells, e.g., T cells, are administered to a subject who has received a previous stem cell transplant (e.g., an autologous stem cell transplant), or a subject who has received a previous dose of melphalan.

[0100] In one embodiment, a cell expressing a CAR molecule, e.g., a CAR molecule described herein, is administered in combination with an agent, e.g., an agent described herein, that ameliorates one or more side effects associated with the administration of a cell expressing a CAR molecule or with the administration of a cytostatic agent.

[0101] In one embodiment, cells expressing a CAR molecule, e.g., a CD19 CAR molecule described herein, and a B cell inhibitor are administered in combination with an additional agent that treats a disease associated with CD19, e.g., an additional agent described herein.

[0102] In one embodiment, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered at a dose and / or dose regimen described herein.

[0103] In one embodiment, CAR molecules are introduced into T cells, for example, using in vitro transcription, and subject (for example, people) accepts the initial administration of cells comprising CAR molecules, and one or more subsequent administrations of cells comprising CAR molecules, wherein the one or more subsequent administrations are less than 15 days after previous administration, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days of administration. In one embodiment, more than once a week, cells comprising CAR molecules are administered to subject (for example, people), for example, 2, 3 or 4 times a week, comprising cells of CAR molecules. In one embodiment, subject (for example, human subjects) accepts more than once a week, comprising cells of CAR molecules, and administers (for example, 2, 3 or 4 times a week) (also referred to herein as cycle), followed by not administering cells comprising CAR molecules for a week, then administering one or more additional administrations of cells comprising CAR molecules to subject (for example, administering cells comprising CAR molecules more than once a week). In another embodiment, the subject (e.g., a human subject) receives more than one cycle of cells comprising CAR molecules, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4 or 3 days. In one embodiment, the cells comprising CAR molecules are administered every other day and 3 times a week. In one embodiment, the cells comprising CAR molecules are administered for at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks or more weeks.

[0104] In one embodiment, a therapy as described herein (e.g., a CD20 CAR therapy, a CD22 CAR therapy or a B cell inhibitor and a CD19 CAR molecule, such as a combination of cells expressing CD19 CAR molecules as described herein) is administered as a disease, such as cancer, for example, a first-line treatment for a cancer as described herein. In another embodiment, a therapy as described herein (e.g., a CD20 CAR therapy, a CD22 CAR therapy or a B cell inhibitor and a CD19 CAR molecule, such as a combination of cells expressing CD19 CAR molecules as described herein) is administered as a disease, such as cancer, for example, a second, third, or fourth-line treatment for a cancer as described herein.

[0105] In one embodiment, a cell population described herein is administered. In some embodiments, a cell population is isolated or purified.

[0106] In one embodiment, the method includes administering a group of cells, wherein a plurality of cells include CAR molecules as described herein. In some embodiments, the colony of CAR-expressing cells includes a mixture of cells expressing different CARs. For example, in one embodiment, the colony of CAR-expressing cells may include a first cell expressing a CAR with an anti-CD19 binding domain as described herein and a second cell expressing a CAR with different B cell antigen binding domains. In embodiments, the first and second cell colonies are T cells. In embodiments, the first and second colonies of T cells are identical isotypes, for example, CD4+T cells, or both are CD8+T cells. In other embodiments, the first and second colonies of T cells are different isotypes, for example, the first colony includes CD4+T cells, and the second colony includes CD8+T cells. In embodiments, the first and second colonies of T cells are cell types described in WO2012 / 129514, the entire contents of which are incorporated herein by reference. As another example, a cell colony may include a single cell type expressing a CAR with an anti-CD19 binding domain as described herein and a CAR with different B cell antigen binding domains. As another example, the cell colony can include a single cell type expressing a CAR having two or more (e.g., 2, 3, 4, or 5) B cell antigen binding domains, for example, a bispecific CAR, for example, as described herein. As another example, a colony of CAR-expressing cells may include a first cell expressing CAR, the CAR including an anti-CD19 binding domain, for example, as described herein, and a second cell expressing CAR, the CAR including an antigen binding domain for a target other than CD19 (e.g., CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, CD79a, or mesothelin). In one embodiment, the colony of CAR-expressing cells includes, for example, a first cell expressing a CAR comprising a major intracellular signaling domain and a second cell expressing a CAR comprising a secondary signaling domain. In one embodiment, the colony of CAR-expressing cells includes, for example, a first cell expressing a CAR comprising a first secondary signaling domain and a second cell expressing CAR, the CAR including a secondary signaling domain different from the first secondary signaling domain.

[0107] For example, when the first B cell inhibitor is a CD19 CAR expressing cell and the second B cell inhibitor is a CD10 CAR expressing cell, the first CAR and the second CAR may be expressed by the same cell type or different types. For example, in some embodiments, the cell expressing CD19 CAR is a CD4+ T cell, the cell expressing CD10 CAR is a CD8+ T cell, or the cell expressing CD19 CAR is a CD8+ T cell, and the cell expressing CD10 CAR is a CD4+ T cell. In other embodiments, the cell expressing CD19 CAR is a T cell, the cell expressing CD10 CAR is a NK cell, or the cell expressing CD19 CAR is a NK cell, and the cell expressing CD10 CAR is a T cell. In other embodiments, the cell expressing CD19 CAR and the cell expressing CD10 CAR are both NK cells, or are both T cells, for example, are both CD4+ T cells, or are both CD8+ T cells. In other embodiments, a single cell expresses CD19 CAR and CD10 CAR, and the cell is, for example, an NK cell or a T cell such as a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR may comprise the same or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR comprises a CD3 ζ signaling domain, and the CD10 CAR comprises a costimulatory domain, such as a 41BB, CD27 or CD28 costimulatory domain, and in some embodiments, the CD19 CAR comprises a costimulatory domain, such as a 41BB, CD27 or CD28 costimulatory domain, and the CD10 CAR comprises a CD3 ζ signaling domain. In other embodiments, each of the CD19 CAR and the CD10 CAR comprises the same type of primary signaling domain, e.g., a CD3ζ signaling domain, but the CD19 CAR and the CD10 CAR comprise different costimulatory domains, e.g., a CD27 costimulatory domain, (2) the CD19 CAR comprises a CD27 costimulatory domain and the CD10 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain (3) the CD19 CAR comprises a 41BB costimulatory domain and the CD10 CAR comprises a CD28 costimulatory domain, (4) the CD19 CAR comprises a CD28 costimulatory domain and the CD10 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (5) the CD19 CAR comprises a CD27 costimulatory domain and the CD10 CAR comprises a CD28 costimulatory domain, or (6) the CD19 The CAR contains the CD28 costimulatory domain, and the CD10 CAR contains the CD27 costimulatory domain.In another embodiment, the cell comprises a CAR comprising a CD19 antigen binding domain and a CD10 antigen binding domain, such as a bispecific antibody.

[0108] As another example, when the first B cell inhibitor is a CD19 CAR expressing cell and the second B cell inhibitor is a CD20 CAR expressing cell, the first CAR and the second CAR can be expressed by the same cell type or different types. For example, in some embodiments, the cells expressing CD19 CAR are CD4+ T cells, the cells expressing CD20 CAR are CD8+ T cells, or the cells expressing CD19 CAR are CD8+ T cells, and the cells expressing CD20 CAR are CD4+ T cells. In other embodiments, the cells expressing CD19 CAR are T cells, the cells expressing CD20 CAR are NK cells, or the cells expressing CD19 CAR are NK cells, and the cells expressing CD20 CAR are T cells. In other embodiments, the cells expressing CD19 CAR and the cells expressing CD20 CAR are both NK cells or both T cells, for example, both are CD4+ T cells, or both are CD8+ T cells. In other embodiments, a single cell expresses CD19 CAR and CD20 CAR, and the cell is, for example, a NK cell or a T cell, such as a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR may comprise the same or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR comprises a CD3ζ signaling domain, and the CD20 CAR comprises a costimulatory domain, such as a 41BB, CD27 or CD28 costimulatory domain, and in some embodiments, the CD19 CAR comprises a costimulatory domain, such as a 41BB, CD27 or CD28 costimulatory domain, and the CD20 CAR comprises a CD3ζ signaling domain. In other embodiments, each of the CD19 CAR and the CD20 CAR comprises the same type of primary signaling domain, e.g., a CD3ζ signaling domain, but the CD19 CAR and the CD20 CAR comprise different costimulatory domains, e.g., (1) the CD19 CAR comprises a 41BB costimulatory domain, and the CD20 CAR comprises a different costimulatory domain, e.g., a CD27 costimulatory domain, (2) the CD19 CAR comprises a CD27 costimulatory domain, and the CD20 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain (3) the CD19 CAR comprises a 41BB costimulatory domain, and the CD20 CAR comprises a CD28 costimulatory domain, (4) the CD19 CAR comprises a CD28 costimulatory domain, and the CD20 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (5) the CD19 CAR comprises a CD27 costimulatory domain, and the CD20 The CAR comprises a CD28 costimulatory domain, or (6) the CD19 CAR comprises a CD28 costimulatory domain and the CD20 CAR comprises a CD27 costimulatory domain.In another embodiment, the cell comprises a CAR comprising a CD19 antigen binding domain and a CD20 antigen binding domain, e.g., a bispecific antibody.

[0109] As another example, when the first B cell inhibitor is a CD19 CAR expressing cell and the second B cell inhibitor is a CD22 CAR expressing cell, the first CAR and the second CAR can be expressed by the same cell type or different types. For example, in some embodiments, the cells expressing CD19 CAR are CD4+ T cells, the cells expressing CD22 CAR are CD8+ T cells, or the cells expressing CD19 CAR are CD8+ T cells, and the cells expressing CD22 CAR are CD4+ T cells. In other embodiments, the cells expressing CD19 CAR are T cells, the cells expressing CD22 CAR are NK cells, or the cells expressing CD19 CAR are NK cells, and the cells expressing CD22 CAR are T cells. In other embodiments, the cells expressing CD19 CAR and the cells expressing CD22 CAR are both NK cells or both T cells, for example, both are CD4+ T cells, or both are CD8+ T cells. In other embodiments, a single cell expresses CD19 CAR and CD22 CAR, and the cell is, for example, an NK cell or a T cell, such as a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR may comprise the same or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR comprises a CD3ζ signaling domain, and the CD22 CAR comprises a costimulatory domain, such as a 41BB, CD27 or CD28 costimulatory domain, and in some embodiments, the CD19 CAR comprises a costimulatory domain, such as a 41BB, CD27 or CD28 costimulatory domain, and the CD22 CAR comprises a CD3ζ signaling domain. In other embodiments, each of the CD19 CAR and the CD22 CAR comprises the same type of primary signaling domain, e.g., a CD3ζ signaling domain, but the CD19 CAR and the CD22 CAR comprise different costimulatory domains, e.g., (1) the CD19 CAR comprises a 41BB costimulatory domain, and the CD22 CAR comprises a different costimulatory domain, e.g., a CD27 costimulatory domain, (2) the CD19 CAR comprises a CD27 costimulatory domain, and the CD22 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (3) the CD19 CAR comprises a 41BB costimulatory domain, and the CD22 CAR comprises a CD28 costimulatory domain, (4) the CD19 CAR comprises a CD28 costimulatory domain, and the CD22 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (5) the CD19 CAR comprises a CD27 costimulatory domain, and the CD22 The CAR contains a CD28 costimulatory domain, or (6) the CD19 CAR contains a CD28 costimulatory domain, and the CD22 CAR contains a CD27 costimulatory domain.In another embodiment, the cell comprises a CAR comprising a CD19 antigen binding domain and a CD22 antigen binding domain, e.g., a bispecific antibody.

[0110] As another example, when the first B cell inhibitor is a CD19 CAR expressing cell and the second B cell inhibitor is a CD34 CAR expressing cell, the first CAR and the second CAR can be expressed by the same cell type or different types. For example, in some embodiments, the cells expressing CD19 CAR are CD4+ T cells, the cells expressing CD34 CAR are CD8+ T cells, or the cells expressing CD19 CAR are CD8+ T cells, and the cells expressing CD34 CAR are CD4+ T cells. In other embodiments, the cells expressing CD19 CAR are T cells, the cells expressing CD34 CAR are NK cells, or the cells expressing CD19 CAR are NK cells, and the cells expressing CD34 CAR are T cells. In other embodiments, the cells expressing CD19 CAR and the cells expressing CD34 CAR are both NK cells or both T cells, for example, both are CD4+ T cells, or both are CD8+ T cells. In other embodiments, a single cell expresses CD19 CAR and CD34 CAR, and the cell is, for example, a NK cell or a T cell, such as a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR may comprise the same or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR comprises a CD3ζ signaling domain, and the CD34 CAR comprises a costimulatory domain, such as a 41BB, CD27 or CD28 costimulatory domain, and in some embodiments, the CD19 CAR comprises a costimulatory domain, such as a 41BB, CD27 or CD28 costimulatory domain, and the CD34 CAR comprises a CD3ζ signaling domain. In other embodiments, each of the CD19 CAR and the CD34 CAR comprises the same type of primary signaling domain, e.g., a CD3ζ signaling domain, but the CD19 CAR and the CD34 CAR comprise different costimulatory domains, e.g., (1) the CD19 CAR comprises a 41BB costimulatory domain, and the CD34 CAR comprises a different costimulatory domain, e.g., a CD27 costimulatory domain, (2) the CD19 CAR comprises a CD27 costimulatory domain, and the CD34 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (3) the CD19 CAR comprises a 41BB costimulatory domain, and the CD34 CAR comprises a CD28 costimulatory domain, (4) the CD19 CAR comprises a CD28 costimulatory domain, and the CD34 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (5) the CD19 CAR comprises a CD27 costimulatory domain, and the CD34 The CAR contains a CD28 costimulatory domain, or (6) the CD19 CAR contains a CD28 costimulatory domain, and the CD34 CAR contains a CD27 costimulatory domain.In another embodiment, the cell comprises a CAR comprising a CD19 antigen binding domain and a CD34 antigen binding domain, e.g., a bispecific antibody.

[0111] As another example, when the first B cell inhibitor is a CD19 CAR expressing cell and the second B cell inhibitor is a CD123 CAR expressing cell, the first CAR and the second CAR can be expressed by the same cell type or different types. For example, in some embodiments, the cells expressing CD19 CAR are CD4+T cells, the cells expressing CD123 CAR are CD8+T cells, or the cells expressing CD19 CAR are CD8+T cells, and the cells expressing CD123 CAR are CD4+T cells. In other embodiments, the cells expressing CD19 CAR are T cells, the cells expressing CD123 CAR are NK cells, or the cells expressing CD19 CAR are NK cells, and the cells expressing CD123 CAR are T cells. In other embodiments, the cells expressing CD19 CAR and the cells expressing CD123 CAR are both NK cells or both T cells, for example, both are CD4+T cells, or both are CD8+T cells. In other embodiments, a single cell expresses CD19 CAR and CD123 CAR, and the cell is, for example, an NK cell or a T cell, such as a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR can comprise the same or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR comprises a CD3 ζ signaling domain, and the CD123 CAR comprises a costimulatory domain, such as 41BB, CD27 or CD28 costimulatory domain, and in some embodiments, the CD19 CAR comprises a costimulatory domain, such as 41BB, CD27 or CD28 costimulatory domain, and the CD123 CAR comprises a CD3 ζ signaling domain.In other embodiments, each of the CD19 CAR and the CD123 CAR comprises the same type of primary signaling domain, e.g., a CD3ζ signaling domain, but the CD19 CAR and the CD123 CAR comprise different costimulatory domains, e.g., a CD27 costimulatory domain, (2) the CD19 CAR comprises a CD27 costimulatory domain and the CD123 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (3) the CD19 CAR comprises a 41BB costimulatory domain and the CD123 CAR comprises a CD28 costimulatory domain, (4) the CD19 CAR comprises a CD28 costimulatory domain and the CD123 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (5) the CD19 CAR comprises a CD27 costimulatory domain and the CD123 CAR comprises a CD28 costimulatory domain, or (6) the CD19 CAR comprises CD28 costimulatory domain, and CD123 CAR comprises CD27 costimulatory domain.In another embodiment, cell comprises CAR, and it comprises CD19 antigen-binding domain and CD123 antigen-binding domain, such as bispecific antibody.

[0112] As another example, when the first B cell inhibitor is a CD19 CAR expressing cell and the second B cell inhibitor is a FLT-3 CAR expressing cell, the first CAR and the second CAR can be expressed by the same cell type or different types. For example, in some embodiments, the cells expressing CD19 CAR are CD4+ T cells, the cells expressing FLT-3 CAR are CD8+ T cells, or the cells expressing CD19 CAR are CD8+ T cells, and the cells expressing FLT-3 CAR are CD4+ T cells. In other embodiments, the cells expressing CD19 CAR are T cells, the cells expressing FLT-3 CAR are NK cells, or the cells expressing CD19 CAR are NK cells, and the cells expressing FLT-3 CAR are T cells. In other embodiments, the cells expressing CD19 CAR and the cells expressing FLT-3 CAR are both NK cells or both T cells, for example, both CD4+ T cells, or both CD8+ T cells. In other embodiments, a single cell expresses CD19 CAR and FLT-3 CAR, and the cell is, for example, a NK cell or a T cell, such as a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR can comprise the same or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR comprises a CD3 ζ signaling domain, and the FLT-3 CAR comprises a costimulatory domain, such as a 41BB, CD27 or CD28 costimulatory domain, and in some embodiments, the CD19 CAR comprises a costimulatory domain, such as a 41BB, CD27 or CD28 costimulatory domain, and the FLT-3 CAR comprises a CD3 ζ signaling domain. In other embodiments, each of the CD19 CAR and the FLT-3 CAR comprises the same type of primary signaling domain, e.g., a CD3ζ signaling domain, but the CD19 CAR and the FLT-3 CAR comprise different costimulatory domains, e.g., (1) the CD19 CAR comprises a 41BB costimulatory domain, and the FLT-3 CAR comprises a different costimulatory domain, e.g., a CD27 costimulatory domain, (2) the CD19 CAR comprises a CD27 costimulatory domain, and the FLT-3 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (3) the CD19 CAR comprises a 41BB costimulatory domain, and the FLT-3 CAR comprises a CD28 costimulatory domain, (4) the CD19 CAR comprises a CD28 costimulatory domain, and the FLT-3 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (5) the CD19 The CAR comprises a CD27 co-stimulatory domain and the FLT-3 CAR comprises a CD28 co-stimulatory domain, or (6) the CD19 CAR comprises a CD28 co-stimulatory domain and the FLT-3 CAR comprises a CD27 co-stimulatory domain.In another embodiment, the cell comprises a CAR comprising a CD19 antigen binding domain and a FLT-3 antigen binding domain, such as a bispecific antibody.

[0113] As another example, when the first B cell inhibitor is a CD19 CAR-expressing cell and the second B cell inhibitor is a ROR1 CAR-expressing cell, the first CAR and the second CAR can be expressed by the same cell type or different types. For example, in some embodiments, the cells expressing CD19 CAR are CD4+ T cells, the cells expressing ROR1 CAR are CD8+ T cells, or the cells expressing CD19 CAR are CD8+ T cells, and the cells expressing ROR1 CAR are CD4+ T cells. In other embodiments, the cells expressing CD19 CAR are T cells, the cells expressing ROR1 CAR are NK cells, or the cells expressing CD19 CAR are NK cells, and the cells expressing ROR1 CAR are T cells. In other embodiments, the cells expressing CD19 CAR and the cells expressing ROR1 CAR are both NK cells or both T cells, for example, both are CD4+ T cells, or both are CD8+ T cells. In other embodiments, a single cell expresses a CD19 CAR and a ROR1 CAR, and the cell is, for example, a NK cell or a T cell, such as a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR may comprise the same or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR comprises a CD3ζ signaling domain, and the ROR1 CAR comprises a costimulatory domain, such as a 41BB, CD27, or CD28 costimulatory domain, and in some embodiments, the CD19 CAR comprises a costimulatory domain, such as a 41BB, CD27, or CD28 costimulatory domain, and the ROR1 CAR comprises a CD3ζ signaling domain. In other embodiments, each of the CD19 CAR and the ROR1 CAR comprises the same type of primary signaling domain, e.g., a CD3ζ signaling domain, but the CD19 CAR and the ROR1 CAR comprise different costimulatory domains, e.g., (1) the CD19 CAR comprises a 41BB costimulatory domain, and the ROR1 CAR comprises a different costimulatory domain, e.g., a CD27 costimulatory domain, (2) the CD19 CAR comprises a CD27 costimulatory domain, and the ROR1 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (3) the CD19 CAR comprises a 41BB costimulatory domain, and the ROR1 CAR comprises a CD28 costimulatory domain, (4) the CD19 CAR comprises a CD28 costimulatory domain, and the ROR1 CAR comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (5) the CD19 CAR comprises a CD27 costimulatory domain, and the ROR1 The CAR contains a CD28 costimulatory domain, or (6) the CD19 CAR contains a CD28 costimulatory domain, and the ROR1 CAR contains a CD27 costimulatory domain.In another embodiment, the cell comprises a CAR comprising a CD19 antigen binding domain and a ROR1 antigen binding domain, such as a bispecific antibody.

[0114] More generally, when the first B cell inhibitor comprises a CD19 CAR and there is a second B cell inhibitor, such as a second B cell inhibitor comprising a second CAR, the first CAR and the second B cell inhibitor can be expressed by the same cell type or different species. For example, in some embodiments, the cells expressing CD19 CAR are CD4+ T cells, and the cells expressing the second B cell inhibitor are CD8+ T cells, or the cells expressing 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 CD19 CAR are T cells, the cells expressing the second B cell inhibitor are NK cells, or the cells expressing CD19 CAR are NK cells, and the cells expressing the second B cell inhibitor are T cells. In other embodiments, the cells expressing 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 other embodiments, a single cell expresses CD19 CAR and a second B cell inhibitor, and the cell is, for example, an NK cell or a T cell such as a CD4+ T cell or a CD8+ T cell. The first CAR and the second CAR may comprise the same or different intracellular signaling domains. For example, in some embodiments, the CD19 CAR comprises a CD3 ζ signaling domain and a second B cell inhibitor (or CAR), comprising a costimulatory domain, such as 41BB, CD27 or CD28 costimulatory domain, and in some embodiments, the CD19 CAR comprises a costimulatory domain, such as 41BB, CD27 or CD28 costimulatory domain and a second B cell inhibitor (or second CAR) comprises a CD3 ζ signaling domain.In other embodiments, each of the CD19 CAR and the second B cell inhibitor (or the second CAR) comprises the same type of primary signaling domain, e.g., a CD3ζ signaling domain, but the CD19 CAR and the second B cell inhibitor comprise different costimulatory domains, e.g., a CD27 costimulatory domain, (2) the CD19 CAR comprises a CD27 costimulatory domain and the second B cell inhibitor (or the second CAR) comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (3) the CD19 CAR comprises a 41BB costimulatory domain and the second B cell inhibitor (or the second CAR) includes a CD28 costimulatory domain, (4) the CD19 CAR comprises a CD28 costimulatory domain and the second B cell inhibitor (or the second CAR) comprises a different costimulatory domain, e.g., a 41BB costimulatory domain, (5) the CD19 CAR comprises CD27 costimulatory domain and the second B cell inhibitor (or the second CAR) comprises CD28 costimulatory domain, or (6) CD19 CAR comprises CD28 costimulatory domain and the second B cell inhibitor (or the second CAR) comprises CD27 costimulatory domain. In another embodiment, the cell comprises CAR, the CAR comprises CD19 antigen binding domain and an antigen binding domain for a second antigen, such as a bispecific antibody.

[0115] In one embodiment, the 4-1BB costimulatory domain comprises the sequence of SEQ ID NO: 16. In one embodiment, the 4-1BB costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 16, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 16. In one embodiment, the 4-1BB costimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO: 60, or a sequence with 95-99% identity thereof.

[0116] In one embodiment, the CD27 costimulatory domain comprises the sequence of SEQ ID NO: 16. In one embodiment, the CD27 costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 16, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 16. In one embodiment, the CD27 costimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO: 17, or a sequence with 95-99% identity thereof.

[0117] In one embodiment, the CD28 costimulatory domain comprises the sequence of SEQ ID NO: 1317. In one embodiment, the CD28 costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 1317, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 1317. In one embodiment, the CD28 costimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO: 1318, or a sequence with 95-99% identity thereof.

[0118] In one embodiment, the wild-type ICOS costimulatory domain comprises the sequence of SEQ ID NO: 1319. In one embodiment, the wild-type ICOS costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 1319, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 1319. In one embodiment, the wild-type ICOS costimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO: 1320, or a sequence with 95-99% identity thereof.

[0119] In one embodiment, the Y to F mutant ICOS costimulatory domain comprises the sequence of SEQ ID NO: 1321. In one embodiment, the Y to F mutant ICOS costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 1321, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 1321. In one embodiment, the Y to F mutant ICOS costimulatory domain is encoded by a nucleic acid sequence with 95-99% identity to the nucleic acid sequence of SEQ ID NO: 1320 (wherein SEQ ID NO: 1320 encodes wild-type ICOS).

[0120] In an embodiment, the primary signaling domain comprises a functional signaling domain of CD3 zeta. In an embodiment, the functional signaling domain of CD3 zeta comprises SEQ ID NO: 17 (mutant CD3 zeta) or SEQ ID NO: 43 (wild-type human CD3 zeta).

[0121] In one embodiment, the method includes administering a cell colony, wherein at least one cell in the colony expresses CAR, such as a CAR with an anti-CD19 domain as described herein, and an agent that enhances the activity of CAR-expressing cells, for example, a second cell expressing an agent that enhances the activity of CAR-expressing cells. For example, in one embodiment, the agent 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 TGFRβ. In one embodiment, the agent that inhibits immunosuppressive molecules includes a first polypeptide, such as an inhibitory molecule, which is combined with a second polypeptide that provides a positive signal to the cell, such as an intracellular signaling domain as described herein. In one embodiment, the agent comprises a first polypeptide, e.g., an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFRβ, 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, the second polypeptide being an intracellular signaling domain described herein (e.g., including a costimulatory domain (e.g., 41BB, CD27 or CD28, e.g., described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent 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 of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein).

[0122] In one embodiment, the B cell inhibitor comprises an inhibitor of one or more 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 that express a CAR molecule that binds to CD10, CD20, CD22, CD34, FLT-3, ROR1, CD79b, CD179b, or CD79a. In one embodiment, the B cell inhibitor comprises CD123 CAR. In one embodiment, the B cell inhibitor comprises one or more cells that express a CAR molecule that binds CD123. In one embodiment, the disease is CD19 negative cancer, such as CD19 negative recurrent cancer. In one embodiment, CD19 CAR expressing cells are administered simultaneously with one or more B cell inhibitors, before or after administration.

[0123] In one embodiment, the method further includes administering a CD19 inhibitor, such as a cell expressing CD19 CAR. In one embodiment, the CD19 inhibitor includes CD19 CAR, and the B cell inhibitor includes CD123 CAR. In one embodiment, CD19 CAR or CD123 CAR include a split intracellular signaling domain so that compared to the activation when CD19 CAR and CD123 CAR are bound to a target cell expressing one of CD19 or CD123 (for example, hematopoietic stem cell), when CD19 CAR and CD123 CAR are both bound to target cells, such as target CD19+CD123+ cells (for example, B-ALL embryonic cells), complete activation of cells such as immune effector cell groups occurs. In one embodiment, CD123 CAR includes 4-1BB signaling domains, and CD19 CAR includes CD3 ζ signaling domains. In one embodiment, CD123 CAR includes a costimulatory domain, such as a 4-1BB signaling domain, and CD19 CAR includes a primary signaling domain, such as a CD3 ζ signaling domain. In one embodiment, CD123CAR includes a primary signaling domain, such as CD3ζ signaling domain, and CD19CAR includes a costimulatory domain, such as a 4-1BB signaling domain. In one embodiment, a B cell inhibitor includes CAR (for example, for CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a CAR), which includes a costimulatory domain, and CD19CAR includes a primary signaling domain. In one embodiment, a B cell inhibitor includes CAR (for example, for CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a CAR), which includes a primary signaling domain, and CD19CAR includes a costimulatory domain. In one embodiment, a B cell inhibitor includes one or more cells expressing a CAR molecule binding CD123, and wherein CD19 CAR expressing cells are administered simultaneously with a B cell inhibitor. In one embodiment, the CD123 CAR comprises a 4-1BB signaling domain and the CD19 CAR comprises a CD3 zeta signaling domain.

[0124] In one embodiment, the method further comprises transplanting cells, such as hematopoietic stem cells or bone marrow, into the mammal.

[0125] On the other hand, the present invention relates to a cell expressing a CAR molecule as described herein, such as a CD19 CAR molecule, in combination with a B cell inhibitor, such as a B cell inhibitor as described herein for use as a drug. On the other hand, the present invention relates to a B cell inhibitor as described herein and a cell expressing a CAR molecule as described herein, such as a CD19 CAR molecule, for use as a drug.

[0126] In another aspect, the present invention relates to a cell expressing a CAR molecule as described herein, e.g., a CD19 CAR molecule, used in combination with a B cell inhibitor, e.g., a B cell inhibitor as described herein, for treating a disease expressing CD19. In another aspect, the present invention relates to a B cell inhibitor as described herein, used in combination with a cell expressing a CAR molecule as described herein, e.g., a CD19 CAR molecule, for treating a disease expressing CD19. In another aspect, the present invention relates to a cell expressing a CAR molecule as described herein, e.g., a CD19 CAR molecule, used in combination with a B cell inhibitor, e.g., a B cell inhibitor as described herein, for treating a cancer, e.g., a cancer described herein.

[0127] In one embodiment, the method includes administering a cell colony, wherein at least one cell in the colony expresses the therapy herein (e.g., CD20 CAR, CD22 CAR or CAR with anti-CD19 domains as described herein in combination with a B cell inhibitor) and an agent that enhances the activity of 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 CAR-expressing cells, for example, simultaneously or soon thereafter. Alternatively, cytokines can be delivered after administering CAR-expressing cells, for example, after assessing the response of the subject to CAR-expressing cells, after an extended period of time. Also provided are related compositions for use and methods for preparing medicines.

[0128] In one embodiment, a cell described herein (e.g., a cell expressing a CD20 CAR molecule, a cell expressing a CD22 CAR molecule, or a cell expressing a CD19 CAR molecule, e.g., a CD19 CAR molecule described herein, in combination with a B cell inhibitor) is administered in combination with an agent that increases the efficacy of the cell expressing the CAR molecule or the inhibitor (e.g., an agent described herein).

[0129] In one embodiment, a cell described herein (e.g., a cell expressing a CD20 CAR molecule, a cell expressing a CD22 CAR molecule, or a cell expressing a CD19 CAR molecule, e.g., a combination of a cell expressing a CD19 CAR molecule described herein and a B cell inhibitor) is administered in combination with an agent (e.g., an agent described herein) that ameliorates one or more side effects associated with the administration of one of the cells expressing the CAR molecule or the inhibitor.

[0130] In one embodiment, cells expressing a CD19 CAR molecule, e.g., a CD19 CAR molecule described herein, are administered in combination with a B cell inhibitor and an agent for treating Hodgkin lymphoma, e.g., an agent described herein.

[0131] In some aspects, the present disclosure provides a method for treating a non-responder, partial responder or relapser of a CD19 inhibitor (e.g., CD19 CAR therapy), including administering a B cell inhibitor to the patient, e.g., a B cell inhibitor as described herein, e.g., CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or all) of CD79a. In embodiments, the B cell inhibitor is a CAR expressing cell (e.g., T cell or NK cell) that is an inhibitor of CD10, CD20, CD22, CD34, CD123, FLT-3, or one or more (e.g., 2, 3, 4, 5, 6 or all) of ROR1. In embodiments, the patient has or is identified as having CD19 negative cancer cells and cancer cells that are positive for one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or all) of 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 to which the cancer cells are positive, e.g., an inhibitor of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or all) of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a to which the cancer cells are positive. In embodiments, the method further comprises determining whether the patient comprises one or both of the steps of CD19 negative cancer cells, and determining whether the patient comprises for CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or all) positive cancer cells. In embodiments, the subject has or is identified as having a tumor or cancer cell colony that is negative for CD19 expression, such as measured by binding to an anti-CD19 antibody (e.g., an antibody with the same specificity as any CAR molecule in Table 2 or Table 3).

[0132] On the other hand, the present invention is characterized in that a composition comprises a cell expressing a chimeric antigen receptor (CAR) molecule that binds CD19 and a B cell inhibitor, wherein the B cell inhibitor is selected from, for example, CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a, or a combination thereof. CAR expressing cells and B cell inhibitors can be present in a single dosage form, or in two or more dosage forms.

[0133] In one embodiment, the composition is a pharmaceutically acceptable composition.

[0134] In embodiments, a composition (eg, nucleic acid, vector, or cell) disclosed herein is used as a medicament.

[0135] In an embodiment, the compositions disclosed herein are used to treat a disease associated with expression of a B cell antigen (eg, CD 19), such as a B cell leukemia or lymphoma.

[0136] CD19 inhibitors

[0137] In embodiments, the CD19 inhibitor is a small molecule, an antibody, an antibody fragment, or a cell therapy.

[0138] In some embodiments, a CD19 inhibitor (e.g., a cell therapy or an antibody) is administered in combination with or is present in a composition with a B cell inhibitor, such as one or more inhibitors of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0139] In one embodiment, the cell expresses 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 comprising a co-stimulatory domain and / or a primary signaling domain). In one embodiment, the CAR 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 comprising a co-stimulatory domain and / or a primary signaling domain).

[0140] In one embodiment, the CAR molecule comprises an anti-CD19 binding domain comprising one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of an anti-CD19 binding domain described herein and one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of an anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain comprising one or more, e.g., all three LC CDRs and one or more, e.g., all three HC CDRs. In one embodiment, the anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of an anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain having two variable heavy chain regions, each comprising HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the anti-CD19 binding domain comprises a murine light chain variable region described herein (e.g., in Table 3) and / or a murine heavy chain variable region described herein (e.g., in Table 3). In one embodiment, the anti-CD19 binding domain is an scFv comprising a murine light chain and a murine heavy chain having the amino acid sequence of Table 3. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided in Table 3, or a sequence with 95-99% identity to an amino acid sequence of Table 3; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided in Table 3, or a sequence with 95-99% identity to an amino acid sequence of Table 3. In one embodiment, the anti-CD19 binding domain comprises the sequence of SEQ ID NO: 59 or a sequence with 95-99% identity thereof. In one embodiment, the anti-CD19 binding domain is an scFv, and the light chain variable region, e.g., in Table 3, comprising an amino acid sequence described herein, is linked to the heavy chain variable region, e.g., in Table 3, comprising an amino acid sequence described herein, via a linker, e.g., a linker described herein. In one embodiment, the anti-CD19 binding domain comprises a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5 or 6, such as 3 or 4 (SEQ ID NO: 53).The light chain variable region and heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0141] In one embodiment, the CAR molecule comprises a humanized anti-CD19 binding domain comprising one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized anti-CD19 binding domain described herein and one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized anti-CD19 binding domain described herein, e.g., a humanized anti-CD19 binding domain comprising one or more, e.g., all three LC CDRs and one or more, e.g., all three HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized anti-CD19 binding domain described herein, e.g., the humanized anti-CD19 binding domain has two variable heavy chain regions, each variable heavy chain region comprising 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_L25 germline sequence. In one embodiment, the light chain variable region comprises a modification (e.g., a substitution, e.g., a substitution of one or more amino acids found in corresponding positions of the murine light chain variable region of SEQ ID NO: 58, e.g., a substitution at one or more of positions 71 and 87). In one embodiment, the heavy chain variable region comprises one, two, three or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has a modification (e.g., a substitution, e.g., a substitution of one or more amino acids found in the corresponding positions of the mouse heavy chain variable region of SEQ ID NO: 58, e.g., a substitution at one or more positions of positions 71, 73 and 78). In one embodiment, the humanized anti-CD19 binding domain comprises a light chain variable region described herein (e.g., in Table 2) and / or a heavy chain variable region described herein (e.g., in Table 2). In one embodiment, the humanized anti-CD19 binding domain is an scFv comprising a light chain and a heavy chain of the amino acid sequence of Table 2.In one embodiment, the humanized anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided in Table 2, or a sequence with 95-99% identity with an amino acid sequence of Table 2; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided in Table 2, or a sequence with 95-99% identity with an amino acid sequence of Table 2. In one embodiment, the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or a sequence with 95-99% identity thereof. In one embodiment, the humanized anti-CD19 binding domain is an scFv, and the light chain variable region comprising an amino acid sequence, e.g., as described herein, is linked to a heavy chain variable region comprising an amino acid sequence, e.g., as described herein, via a linker, e.g., as described herein. In one embodiment, the humanized anti-CD19 binding domain comprises a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, e.g., 3 or 4 (SEQ ID NO: 53). The light chain variable region and heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0142] In one embodiment, the CAR molecule comprises an anti-CD19 binding domain comprising one or more (e.g., 2, 3, 4, 5 or 6) LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2 and HC CDR3 of a construct of Tables 4 and 5, e.g., murine_CART19, humanized_CART19 a, humanized_CART19 b, or humanized_CART19 c.

[0143] In one embodiment, the CAR molecule comprises a leader sequence, e.g., a leader sequence described herein, e.g., a leader sequence of SEQ ID NO: 13, or a sequence with 95-99% identity thereof; an anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 described herein, e.g., a murine anti-CD19 binding domain described in Table 3, a humanized anti-CD19 binding domain described in Table 2, or a sequence with 95-99% identity thereof; a hinge region, e.g., a hinge region described herein, e.g., a hinge region of SEQ ID NO: 14, or a hinge region with 95-99% identity thereof; a transmembrane domain, e.g., a transmembrane domain described herein, e.g., a transmembrane domain having SEQ ID NO: 15; NO: 15, or a transmembrane domain having a sequence having 95-99% identity thereof; an intracellular signaling domain, e.g., an intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain). In one embodiment, the intracellular signaling domain comprises a costimulatory domain, e.g., a costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having a sequence having SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence having 95-99% identity thereof, and / or a primary signaling domain, e.g., a primary signaling domain described herein, e.g., a CD3 zeta-stimulatory domain having a sequence having SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence having 95-99% identity thereof.

[0144] In one embodiment, the CAR molecule comprises (e.g., consists of) 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, or 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. NO:42, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to 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.

[0145] The present invention generally relates in some aspects to cells engineered to express CAR, such as T cells or natural killer (NK) cells, in combination with one or more B cell inhibitors for the treatment of diseases associated with expression of cluster of differentiation antigens 19 protein (CD19). In some embodiments, the B cell inhibitor is an inhibitor of one or more of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0146] In some embodiments, the CD19 inhibitor comprises an antibody molecule, such as an antibody molecule having a CD19 binding sequence described herein. For example, the antibody molecule may comprise a CDR or VH and VL as described in any one of Tables 2, 3, 4, and 5, or a sequence homologous thereto, such as a sequence having 95-99% identity thereto. The antibody molecule may comprise a CD19 binding region having a sequence described in this section, such as in the context of a CAR.

[0147] In embodiments, the B cell inhibitor is selected from an inhibitory nucleic acid that binds to one or more B cell antigens (e.g., one or more of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a), a soluble ligand, an antibody or antigen-binding fragment thereof, a CAR or a cell expressing a CAR.

[0148] CD20 binding domain and inhibitors

[0149] In some aspects, the present disclosure provides a CD20 inhibitor or binding domain, such as a CD20 inhibitor or binding domain described herein. The present disclosure also provides a nucleic acid encoding a CD20 binding domain, such as a nucleic acid encoding a CAR comprising a CD20 binding domain. The composition may further comprise a second active agent, such as an anti-CD19 CAR expressing cell or a CD19 binding domain. The active agent may be, for example, encoded by a single nucleic acid or different nucleic acids.

[0150] In some aspects, the CD20 inhibitor or binding domain is administered as a monotherapy. In some aspects, the CD20 inhibitor or binding domain is administered in combination with a second active agent, such as an anti-CD19 CAR expressing cell.

[0151] The CD20 inhibitor can be, for example, a small molecule, an antibody or its antigen-binding fragment, a CAR or a CAR-expressing cell. 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, veltuzumab, obinutuzumab, ocaratuzumab or PRO131921 (Genentech). In one embodiment, the CD20 inhibitor is a fusion protein comprising a portion of an anti-CD20 antibody, such as TRU-015 (Trubion Pharmaceuticals).

[0152] In one embodiment, the CD20 inhibitor is an anti-CD20 expressing cell, such as a CD20 CART or a CD20 expressing NK cell.

[0153] In some embodiments, the CD20-CAR comprises an optional leader sequence (e.g., an optional 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 CD20 CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain, a hinge, a transmembrane domain, an intracellular costimulatory domain (e.g., an intracellular costimulatory domain described herein), and an intracellular stimulatory domain.

[0154] In one embodiment, the CD20 binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a CD20 binding domain described herein and / or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a CD20 binding domain described herein, e.g., a CD20 binding domain comprising one or more, e.g., all three LC CDRs and one or more, e.g., all three HC CDRs. These CDRs can be, e.g., those of Tables 12A, 12B, and / or 13. In one embodiment, the CD20 binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a CD20 binding domain described herein, e.g., a CD20 binding domain has two variable heavy chain regions, each comprising HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the CD20 binding domain comprises a light chain variable region described herein (e.g., in Table 15A or 15B) and / or a heavy chain variable region described herein (e.g., in Table 14A or 14B). In one embodiment, the CD20 binding domain comprises a heavy chain variable region described herein (e.g., in Table 14A or 14B), e.g., at least two heavy chain variable regions described herein (e.g., in Table 14A or 14B). In one embodiment, the CD20 binding domain is an scFv comprising a light chain and a heavy chain of the amino acid sequence of Table 14A or 14B or 15A or 15B. In one embodiment, the CD20 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in Table 15A or 15B, or a sequence with 95-99% identity to an amino acid sequence of Table 15A or 15B; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided in Table 14A or 14B, or a sequence with 95-99% identity to an amino acid sequence of Table 14A or 14B. The CD20 binding domain can be, for example, part of an antibody molecule or a CAR molecule.

[0155] In one embodiment, the CAR molecule comprises an anti-CD20 binding domain which includes one or more (e.g., 2, 3, 4, 5 or 6) LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 of a construct 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.

[0156] In one embodiment, the CAR molecule comprises an anti-CD22 binding domain comprising the VL and / or VH of the construct of Tables 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.

[0157] The CD20 scFv can be preceded by an optional leader sequence, e.g., as provided in SEQ ID NO: 13, followed by an optional hinge sequence, e.g., as provided in SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, a transmembrane region, e.g., as provided in SEQ ID NO: 15, an intracellular signaling domain comprising SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3 zeta sequence comprising SEQ ID NO: 17 or SEQ ID NO: 43, e.g., wherein the domains are contiguous and in the same reading frame to form a single fusion protein.

[0158] Additional embodiments include nucleotide sequences encoding the polypeptides of any one of Tables 11A-15B. Other embodiments include nucleotide sequences encoding the polypeptides of any one of Tables 11A-15B, and each of SEQ ID NOs: 13, 14, 15, 16, 17, and optionally 51.

[0159] In one embodiment, the CD20 binding domain is characterized by a specific functional characteristic or property of an antibody or antibody fragment. For example, in one embodiment, the portion of the CAR composition of the invention comprising the antigen binding domain specifically binds to human CD20 or a fragment thereof.

[0160] In one embodiment, the CD20 binding domain is a fragment, such as a single-chain variable fragment (scFv). In one embodiment, the CD20 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 fragments thereof of the present invention bind to the CD20 protein or its fragment with wild-type or enhanced affinity. In some cases, the human scFv can be derived from a display library.

[0161] In one embodiment, the CD20 binding domain, such as an scFv, comprises at least one mutation such that the mutated scFv confers increased stability to the CART20 construct. In another embodiment, the CD20 binding domain, such as an scFv, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations, such as those resulting from a humanization process, such that the mutated scFv confers increased stability to the CART20 construct.

[0162] In some embodiments, the CD20 inhibitor comprises an antibody molecule, such as an antibody molecule having a CD20 binding sequence described herein. For example, the antibody molecule may comprise a CDR or VH and VL as described in any one of Tables 11A-15B, or a sequence homologous thereto, such as a sequence having 95-99% identity thereto. The antibody molecule may comprise a CD20 binding region having a sequence described in this section, such as in the context of a CAR.

[0163] On the one hand, the present disclosure provides a group of CAR expressing cells, such as CART cells, comprising a cell mixture expressing CD19 CAR and CD20 CAR. For example, in one embodiment, the CART cell colony can include a first cell expressing CD19 CAR and a second cell expressing CD20 CAR.

[0164] In some aspects, a 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 to CD20, wherein the second antibody molecule is an antibody molecule described herein, e.g., an antibody molecule selected from Tables 11A-15B. In some embodiments, a binding domain or antibody molecule described herein competes for binding and / or binds to the same (or substantially the same) or overlapping (or substantially overlapping) epitope as a second antibody molecule to CD20, wherein the second antibody molecule is an antibody molecule described herein, e.g., an antibody molecule selected from Tables 11A-15B, e.g., as determined by the methods described in Example 25. In some embodiments, a biparatopic CD20 binding domain binds a first epitope, e.g., an epitope bound by an antibody molecule selected from Tables 11A-15B, and the biparatopic binding domain also binds a second epitope, e.g., a second epitope bound by an antibody molecule selected from Tables 11A-15B. In some aspects, the present disclosure provides a method of treatment comprising administering a first CD20 binding domain that binds to a first epitope (e.g., an epitope bound by an antibody molecule selected from Tables 11A-15B), and a second CD20 binding domain that binds to a second epitope (e.g., a second epitope bound by an antibody molecule selected from Tables 11A-15B). In some embodiments, the CD20 binding domain is part of a CAR molecule, e.g., expressed by a cell expressing a CAR.

[0165] CD22 binding domain and inhibitors

[0166] In some aspects, the present disclosure provides a CD22 inhibitor or binding domain, such as a CD22 inhibitor or binding domain described herein. The present disclosure also provides nucleic acids encoding CD22 binding domains, such as nucleic acids encoding CARs comprising CD22 binding domains. The composition may also include a second active agent, such as an anti-CD19 CAR expressing cell or CD19 binding domain. The active agent may be, for example, encoded by a single nucleic acid or different nucleic acids.

[0167] In some aspects, the CD22 inhibitor or binding domain is administered as a monotherapy. In some aspects, the CD22 inhibitor or binding domain is administered in combination with a second active agent such as an anti-CD19 CAR expressing cell.

[0168] CD22 inhibitors can be, for example, small molecules, antibodies or their antigen-binding fragments, CAR or CAR-expressing cells. In one embodiment, the CD22 inhibitor is an anti-CD22 antibody or its fragment. 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, optionally conjugated to a second active agent 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, such as the scFv of antibody RFB4. The scFv can be fused to all or a fragment of Pseudomonas 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 its fragment comprises the Fv portion of an anti-CD22 antibody, which is optionally covalently fused to all or a fragment (e.g., 38KDa fragment) of Pseudomonas exotoxin-A (e.g., moxetumomab pasudotox). In one embodiment, the anti-CD22 antibody is an anti-CD19 / CD22 bispecific antibody, optionally conjugated to a toxin. For example, in one embodiment, the anti-CD22 antibody comprises an anti-CD19 / CD22 bispecific portion (e.g., two scFv ligands, recognizing human CD19 and CD22) optionally linked to all or part of diphtheria toxin (DT), e.g., the first 389 amino acids of diphtheria toxin (DT), DT 390, e.g., a ligand-directed toxin such as DT2219ARL). In another embodiment, the bispecific portion (e.g., anti-CD19 / anti-CD22) is linked to a toxin such as a deglycosylated ricin A chain (e.g., Combotox).

[0169] In one embodiment, the CD22 inhibitor is an anti-CD22 expressing cell, such as a CD22 CART or a CD22 expressing NK cell.

[0170] On the one hand, the present disclosure provides a group of CAR expressing cells, such as CART cells, comprising a mixture of cells expressing CD19 CAR and CD22 CAR. For example, in one embodiment, the CART cell colony may include a first cell expressing CD19 CAR and a second cell expressing CD22 CAR. As another example, the colony of CAR T cells may include a single colony expressing more than one, such as 2, 3, 4, 5 or 6 or more CARs, such as CD19 CAR and CD22 CAR.

[0171] In some embodiments, the CD22-CAR comprises an optional leader sequence (e.g., an optional 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 CD22 CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain, a hinge, a transmembrane domain, an intracellular costimulatory domain (e.g., an intracellular costimulatory domain described herein), and an intracellular stimulatory domain.

[0172] In one embodiment, the CD22 binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a CD22 binding domain described herein and / or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a CD22 binding domain described herein, e.g., a CD22 binding domain comprising one or more, e.g., all three LC CDRs and one or more, e.g., all three HC CDRs. These CDRs can be, for example, one or more CDRs of Tables 7A, 7B, 7C, 8A, and / or 8B. In one embodiment, the CD22 binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a CD22 binding domain described herein, e.g., a CD22 binding domain having two variable heavy chain regions, each comprising HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the CD22 binding domain comprises 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 comprises a heavy chain variable region described herein (e.g., in Table 9A or 9B), e.g., 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 comprising a light chain and a heavy chain of an amino acid sequence of Table 9A or 9B or 10A or 10B. In one embodiment, the CD22 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in Table 10A or 10B, or a sequence with 95-99% identity to an amino acid sequence of Table 10A or 10B; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided in Table 9A or 9B, or a sequence with 95-99% identity to an amino acid sequence of Table 9A or 9B. The CD22 binding domain can be, for example, part of an antibody molecule or a CAR molecule.

[0173] In one embodiment, the CAR molecule comprises an anti-CD22 binding domain comprising one or more (e.g., 2, 3, 4, 5 or 6) LC CDR1s, LC CDR2s, LC CDR3s, HC CDR1s, HC CDR2s, and HC CDR3s of a construct of Tables 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, CAR22-19 ,CAR22-20,CAR22-21,CAR22-22,CAR22-23,CAR22-24,CAR22-25,CAR22-26,CAR22-27,CAR22-28,CAR2 2-29, CAR22-30, CAR22-31, CAR22-32, CAR22-33, CAR22-34, CAR22-35, CAR22-36, CAR22-37, or CAR22-38.

[0174] In one embodiment, the CAR molecule comprises an anti-CD22 binding domain comprising the VL and / or VH of the construct of Tables 9A, 9B, 10A, and / or 10B, 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 CAR22-35, CAR22-36, CAR22-37, or CAR22-38, or a sequence 95-99% identical thereto.

[0175] The scFv can be preceded by an optional leader sequence, e.g., as provided in SEQ ID NO: 13, followed by an optional hinge sequence, e.g., as provided in SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, a transmembrane region, e.g., as provided in SEQ ID NO: 15, an intracellular signaling domain comprising SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3 zeta sequence comprising SEQ ID NO: 17 or SEQ ID NO: 43, e.g., wherein the domains are adjacent and in the same reading frame to form a single fusion protein.

[0176] Additional embodiments include nucleotide sequences encoding the polypeptides of any one of Tables 6A-10B. Other embodiments include nucleotide sequences encoding the polypeptides of any one of Tables 6A-10B, and each of SEQ ID NOs: 13, 14, 15, 16, 17, and optionally 51.

[0177] In one embodiment, the CD22 binding domain is characterized by a specific functional characteristic or property of an antibody or antibody fragment. For example, in one embodiment, the portion of the CAR composition of the present invention comprising the antigen binding domain specifically binds to human CD22 or a fragment thereof.

[0178] 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 fragments thereof of the present invention bind to the CD22 protein or its fragment with wild-type or enhanced affinity. In some cases, human scFv can be derived from a display library.

[0179] In one embodiment, the CD22 binding domain, such as scFv, comprises at least one mutation such that the mutated scFv confers better stability to the CART22 construct. In another embodiment, the CD22 binding domain, such as scFv, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations, such as those resulting from a humanization process, such that the mutated scFv confers improved stability to the CART22 construct.

[0180] In some embodiments, the CD22 inhibitor comprises an antibody molecule, such as an antibody molecule having a CD22 binding sequence described herein. For example, the antibody molecule may comprise a CDR or VH and VL as described in any one of Tables 6A-10B, or a sequence homologous thereto, such as a sequence having 95-99% identity thereto. The antibody molecule may comprise a CD22 binding region having a sequence described in this section, such as in the context of a CAR.

[0181] In one embodiment, the present disclosure provides a group of CAR expressing cells, such as CART cells, comprising a cell mixture expressing CD19CAR and CD22CAR. For example, in one embodiment, the CART cell colony can include a first cell expressing CD19CAR and a second cell expressing CD22CAR.

[0182] In some aspects, a 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 to CD22, wherein the second antibody molecule is an antibody molecule described herein, e.g., an antibody molecule selected from Tables 6A-10B. In some embodiments, a binding domain or antibody molecule described herein competes for binding and / or binds to the same (or substantially the same) or overlapping (or substantially overlapping) epitope as a second antibody molecule to CD22, wherein the second antibody molecule is an antibody molecule described herein, e.g., an antibody molecule selected from Tables 6A-10B, e.g., as determined by the methods described in Example 25. In some embodiments, a biparatopic CD22 binding domain binds a first epitope, e.g., an epitope bound by an antibody molecule selected from Tables 6A-10B, and the biparatopic binding domain also binds a second epitope, e.g., 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 (e.g., an epitope bound by an antibody molecule selected from Tables 6A-10B), and a second CD22 binding domain that binds to a second epitope (e.g., 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, e.g., expressed by a cell expressing a CAR.

[0183] In some embodiments, the CD22 binding domain binds to one or more of 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; domains 3 and 4; or domains 5, 6, and 7.

[0184] In some aspects, the present disclosure provides a method for treating a CD19 negative cancer, such as a leukemia, such as ALL, such as B-ALL, comprising administering a CD22 inhibitor, such as a CD22 binding domain or a CD22 CAR expressing cell as described herein. In some embodiments, the method includes a step of determining that the cancer is CD19 negative. In some embodiments, the subject has received a CD19 inhibitor, such as a CD19 CAR expressing cell, and is resistant, relapsed, or refractory to the CD19 inhibitor.

[0185] ROR1 inhibitors

[0186] The ROR1 inhibitor can 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, for example, cirmtuzumab.

[0187] In one embodiment, the ROR1 inhibitor is an anti-ROR1 expressing cell, such as a ROR1 CART or a ROR1 expressing NK cell.

[0188] In some embodiments, ROR1-CAR includes an optional leader sequence (e.g., an optional 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 as described herein) and an intracellular stimulation domain (e.g., an intracellular stimulation domain as described herein). In one embodiment, exemplary ROR1 CAR constructs include an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain, a hinge, a transmembrane domain, an intracellular costimulatory domain (e.g., an intracellular costimulatory domain as described herein) and an intracellular stimulation domain.

[0189] In one embodiment, the ROR1 binding domain comprises a scFv portion, e.g., a human scFv portion. The scFv can be preceded by an optional leader sequence, e.g., a leader sequence provided in SEQ ID NO: 13, followed by an optional hinge sequence, e.g., a hinge sequence 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 provided in SEQ ID NO: 15, an intracellular signaling domain comprising SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3 zeta sequence comprising SEQ ID NO: 17 or SEQ ID NO: 43, e.g., wherein the domains are adjacent and in the same reading frame to form a single fusion protein.

[0190] In some embodiments, the present 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, such as described herein, for example, adjacent to and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. Exemplary intracellular signaling domains that can be used in CAR include, but are not limited to, one or more intracellular signaling domains such as CD3-ζ, CD28, 4-1BB, etc. In some cases, CAR can comprise any combination of CD3-ζ, CD28, 4-1BB, etc.

[0191] In one embodiment, the ROR1 binding domain is characterized by a specific functional feature or characteristic of an antibody or antibody fragment. For example, in one embodiment, a portion of the CAR composition of the present invention comprising an antigen binding domain specifically binds to human ROR1 or a fragment thereof. In certain embodiments, the scFv is adjacent to the leader sequence and is in the same open reading frame. In one aspect, the leader sequence is a polypeptide sequence as provided in SEQ ID NO: 13.

[0192] In one embodiment, the ROR1 binding domain is a fragment, such as a single-chain variable fragment (scFv). In one embodiment, the ROR1 binding domain is an Fv, Fab, a(Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the present invention bind to the ROR1 protein or its fragment with wild-type or enhanced affinity. In some cases, human scFv can be derived from a display library.

[0193] In one embodiment, the ROR1 binding domain, such as scFv, comprises at least one mutation such that the mutated scFv confers improved stability to the ROR1 CART construct. In another embodiment, the ROR1 binding domain, such as scFv, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations from the humanization process such that the mutated scFv confers improved stability to the ROR1 CART construct.

[0194] In one embodiment, the present disclosure provides a group of CAR expressing cells, such as CART cells, comprising a mixture of cells expressing CD19CAR and ROR1 CAR. For example, in one embodiment, the CART cell colony can include a first cell expressing CD19 CAR and a second cell expressing ROR1 CAR.

[0195] CD123 inhibitors

[0196] The CD123 inhibitor can be, for example, a small molecule, an antibody or a fragment thereof (e.g., a monospecific or bispecific antibody or a fragment thereof); a recombinant protein that binds to CD123, such as a fusion protein; an inhibitory nucleic acid; or a cell expressing a CD123 CAR, such as a CD123 CART.

[0197] In one embodiment, the CD123 inhibitor is a recombinant protein, eg, comprising the natural ligand (or fragment) of the CD123 receptor, such as SL-401 (also known as DT388IL3, University of Texas Southwestern Medical Center).

[0198] In another embodiment, the CD123 inhibitor is an anti-CD123 antibody or a fragment thereof, such as a monoclonal antibody (e.g., a monospecific or bispecific antibody or a fragment thereof), such as CSL360 (CSL Limited), CSL362 (CSL Limited) or MGD006 (MacroGenics).

[0199] In one embodiment, the CD123 inhibitor is an anti-CD123 CAR-expressing cell, such as a CD123 CART or a CD123 CAR-expressing NK cell.

[0200] In some embodiments, CD123-CAR includes an optional leader sequence (e.g., an optional leader sequence as described herein), an extracellular antigen binding domain, a hinge (e.g., a hinge as described herein), a membrane spaning domain (e.g., a membrane spaning domain as described herein) and an intracellular stimulation domain (e.g., an intracellular stimulation domain as described herein). In one embodiment, exemplary CD123 CAR constructs include an optional leader sequence (e.g., a leader sequence as described herein), an extracellular antigen binding domain, a hinge, a membrane spaning domain, an intracellular costimulatory domain (e.g., an intracellular costimulatory domain as described herein) and an intracellular stimulation domain.

[0201] In one embodiment, the CD123 binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a CD123 binding domain described herein and / or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a CD123 binding domain described herein, e.g., a CD123 binding domain comprising one or more, e.g., all three LC CDRs and one or more, e.g., all three HC CDRs. These CDRs can be, e.g., those of any one of Tables 17, 18, 26, or 27. In one embodiment, the CD123 binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1) of the CD123 binding domain described herein, heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3), e.g., a CD123 binding domain has two variable heavy chain regions, each comprising HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the CD123 binding domain comprises a light chain variable region and / or a heavy chain variable region as described herein. In one embodiment, the CD123 binding domain comprises a heavy chain variable region as described herein, e.g., at least two heavy chain variable regions as described herein. In one embodiment, the CD123 binding domain is an scFv comprising a light chain and a heavy chain of the amino acid sequence of Table 16 or 25. In one embodiment, the CD33 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region in Table 16 or 25, or a sequence with 95-99% identity with a light chain variable region in Table 16 or 25; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region in Table 16 or 25, or a sequence with 95-99% identity with a heavy chain variable region in Table 16 or 25.

[0202] In one embodiment, the CAR molecule includes an anti-CD123 binding domain comprising one or more (e.g., 2, 3, 4, 5 or 6) LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 of the constructs of Tables 17 and 18, such as CAR123-1, CAR123-2, CAR123-3 or CAR123-4. In one embodiment, the CAR molecule includes an anti-CD123 binding domain comprising one or more (e.g., 2, 3, 4, 5 or 6) LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 of the constructs of Tables 26 and 27, such as hzCAR123.

[0203] The CD123 scFv can be preceded by an optional leader sequence, e.g., the leader sequence provided in SEQ ID NO: 13, followed by an optional hinge sequence, e.g., the hinge sequence 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 comprising SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3 zeta sequence comprising SEQ ID NO: 17 or SEQ ID NO: 43, e.g., wherein the domains are adjacent and in the same reading frame to form a single fusion protein.

[0204] Additional embodiments include nucleotide sequences encoding the polypeptides of any one of Tables 16-27. Additional embodiments include nucleotide sequences encoding the polypeptides of any one of Tables 16-27, and each of the domains of SEQ ID NOs: 13, 14, 15, 16, 17, and optionally 51.

[0205] In one embodiment, the CD123 binding domains are characterized by specific functional characteristics or properties of an antibody or antibody fragment. For example, in one embodiment, the part of the CAR composition of the present invention comprising an antigen binding domain specifically binds to human CD123 or its fragment.

[0206] In one embodiment, the CD123 binding domains are fragments, such as single-chain variable fragments (scFv). In one embodiment, the CD123 binding domains are Fv, Fab, (Fab')2 or bifunctional (such as bispecific) hybrid antibodies (for example, Lanzavecchia et al., Eur.J.Immunol.17,105 (1987)). On the one hand, the antibody of the present invention and its fragment are bound to CD123 protein or its fragment with wild type or enhanced affinity. In some cases, human scFv can be from display library.

[0207] In one embodiment, CD123 binding domains such as scFv include at least one mutation so that the scFv of the mutation gives CART123 constructs improved stability. In another embodiment, CD123 binding domains such as scFv include at least 1,2,3,4,5,6,7,8,9,10 mutations, such as mutations generated from a humanization process, so that the scFv of the mutation gives CART123 constructs improved stability.

[0208] In some embodiments, the CD123 inhibitor includes an antibody molecule, for example, an antibody molecule with a CD123 binding sequence as described herein. For example, the antibody molecule may include a CDR or VH and VL as described in any one of Tables 16-27, or a sequence homologous thereto, for example, a sequence with 95-99% identity thereto. The antibody molecule may include a CD123 binding region having a sequence such as described in this section in the context of CAR.

[0209] In one embodiment, the disclosure provides a group of CAR expressing cells, such as CART cells, which include a cell mixture expressing CD19CAR and CD123CAR. For example, in one embodiment, CART cell colonies can include a first cell expressing CD19CAR and a second cell expressing CD123CAR.

[0210] CD10 inhibitors

[0211] The CD10 inhibitor can be, for example, a small molecule, an antibody or fragment thereof (e.g., a monospecific or bispecific antibody or fragment thereof); a recombinant protein that binds to CD10, such as a fusion protein; an inhibitory nucleic acid; or a cell expressing a CD10 CAR, such as a CD10 CART.

[0212] In one embodiment, the CD10 inhibitor comprises a small molecule, such as sacubitril (Novartis), valsartan / sacubritril (Novartis), omapatrilat (Bristol-Myers Squibb), RB-101, UK-414,495 (Pfizer), or a pharmaceutically acceptable salt or derivative thereof.

[0213] In one embodiment, the CD10 inhibitor is an anti-CD10 CAR-expressing cell, such as a CD10 CART or a CD10 CAR-expressing NK cell.

[0214] In one embodiment, the present disclosure provides a group of CAR expressing cells, such as CART cells, comprising a cell mixture expressing CD19CAR and CD10CAR. For example, in one embodiment, the CART cell colony can include a first cell expressing CD19CAR and a second cell expressing CD10CAR.

[0215] CD34 inhibitors

[0216] The CD34 inhibitor can be, for example, a small molecule, an antibody or fragment thereof (e.g., a monospecific or bispecific antibody or fragment thereof); a recombinant protein that binds to CD34, such as a fusion protein; an inhibitory nucleic acid; or a cell expressing a CD34 CAR, such as a CD34 CART.

[0217] In one embodiment, the CD34 inhibitor comprises a monoclonal antibody or a fragment thereof targeting CD34 or an immunoliposome comprising an anti-CD34 monoclonal antibody or a fragment thereof.

[0218] In one embodiment, the CD34 inhibitor is an anti-CD34 CAR-expressing cell, such as a CD34 CART or a CD34 CAR-expressing NK cell.

[0219] In one embodiment, the present disclosure provides a group of CAR expressing cells, such as CART cells, comprising a cell mixture expressing CD19CAR and CD34CAR. For example, in one embodiment, the CART cell colony can include a first cell expressing CD19CAR and a second cell expressing CD34CAR.

[0220] FLT-3 inhibitors

[0221] The FLT-3 inhibitor can be, for example, a small molecule, an antibody or a fragment thereof (e.g., a monospecific or bispecific antibody or a fragment thereof); a recombinant protein that binds to FLT-3, such as a fusion protein; an inhibitory nucleic acid; or a cell expressing FLT-3 CAR, such as a FLT-3 CART.

[0222] In some embodiments, the FLT-3 inhibitor comprises a small molecule, such as quizartinib (Ambit Biosciences), midostaurin (Technische Universitat Dresden), sorafenib (Bayer and Onyx Pharmaceuticals), sunitinib (Pfizer), lestaurtinib (Cephalon), or an acceptable salt or derivative thereof.

[0223] In one embodiment, the FLT-3 inhibitor is an anti-FLT-3 CAR expressing cell, such as a FLT-3 CART or a NK cell expressing FLT-3 CAR.

[0224] In one embodiment, the present disclosure provides a group of CAR expressing cells, such as CART cells, comprising a cell mixture expressing CD19CAR and FLT-3CAR. For example, in one embodiment, the CART cell colony can include a first cell expressing CD19CAR and a second cell expressing FLT-3CAR.

[0225] CD79b inhibitors

[0226] In certain embodiments, cells expressing CD19 CAR are administered together with CD79b inhibitors. CD79b inhibitors can be, for example, small molecules, antibodies or fragments thereof (e.g., monospecific or bispecific antibodies or fragments thereof); recombinant proteins that bind CD79b, such as fusion proteins; inhibitory nucleic acids; or cells expressing CD79b CAR, such as T cells or NK cells expressing CD79b CAR. In one embodiment, the CD79b inhibitor is an anti-CD79b CAR expressing cell, such as CD79b CART or NK cells expressing CD79b CAR. Exemplary CD79b inhibitors are described in more detail below.

[0227] In one embodiment, the present disclosure provides a group of CAR expressing cells, such as CART cells or NK cells expressing CAR, which include a mixture of cells expressing CD19 CAR and CD79b CAR. For example, in one embodiment, the colony of CAR expressing cells includes a first cell expressing CD19 CAR and a second cell expressing CD79b CAR.

[0228] CD179b inhibitors

[0229] In certain embodiments, cells expressing CD19 CAR are administered together with CD179b inhibitors. CD179b inhibitors can be, for example, small molecules, antibodies or fragments thereof (e.g., monospecific or bispecific antibodies or fragments thereof); recombinant proteins that bind CD179b, such as fusion proteins; inhibitory nucleic acids; or cells expressing CD179b CAR, such as T cells or NK cells expressing CD179b CAR. In one embodiment, the CD179b inhibitor is an anti-CD179b CAR expressing cell, such as CD179b CART or NK cells expressing CD179b CAR. Exemplary CD179b inhibitors are described in more detail below.

[0230] In one embodiment, the present disclosure provides a group of CAR expressing cells, such as CART cells or NK cells expressing CAR, which include a mixture of cells expressing CD19 CAR and CD179b CAR. For example, in one embodiment, the colony of CAR expressing cells includes a first cell expressing CD19 CAR and a second cell expressing CD179b CAR.

[0231] CD79a inhibitors

[0232] In certain embodiments, cells expressing CD19 CAR are administered together with CD79a inhibitors. CD79a inhibitors can be, for example, small molecules, antibodies or fragments thereof (e.g., monospecific or bispecific antibodies or fragments thereof); recombinant proteins that bind CD79a, such as fusion proteins; inhibitory nucleic acids; or cells expressing CD79a CAR, such as T cells or NK cells expressing CD79a CAR. In one embodiment, the CD79a inhibitor is an anti-CD79a CAR expressing cell, such as CD79a CART or NK cells expressing CD79a CAR. Exemplary CD79a inhibitors are described in more detail below.

[0233] In one embodiment, the present disclosure provides a group of CAR expressing cells, such as CART cells or NK cells expressing CAR, which include a mixture of cells expressing CD19 CAR and CD79a CAR. For example, in one embodiment, the colony of CAR expressing cells includes a first cell expressing CD19 CAR and a second cell expressing CD79a CAR.

[0234] CAR molecules

[0235] A binding domain described herein (e.g., a binding domain to one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a) can further comprise one or more additional amino acid sequences.

[0236] In one embodiment, the CAR molecule comprises an α, β or ζ chain selected from the group consisting of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the transmembrane domain comprises a sequence of SEQ ID NO: 15. In one embodiment, the transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (eg, substitutions) but no more than 20, 10 or 5 modifications (eg, substitutions) of an amino acid sequence of SEQ ID NO: 15 or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 15.

[0237] In one embodiment, the binding domain is connected to the transmembrane domain via a hinge region (e.g., a hinge region described herein). In one embodiment, the encoded hinge region comprises SEQ ID NO: 14 or SEQ ID NO: 45, or a sequence with 95-99% identity thereof.

[0238] In one embodiment, the CAR molecule further comprises a sequence encoding a costimulatory domain, such as a costimulatory domain as described herein. In one embodiment, the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137). In one embodiment, the costimulatory domain comprises a sequence of SEQ ID NO: 16. In one embodiment, the costimulatory domain comprises a sequence of SEQ ID NO: 51. In one embodiment, the costimulatory domain comprises an amino acid sequence having SEQ ID NO: 16 or SEQ ID NO: 51, at least one, two or three modifications (eg, substitutions) but no more than 20, 10 or 5 modifications (eg, substitutions), or a sequence with SEQ ID NO: 16 or SEQ ID NO: 51 amino acid sequence having 95-99% identity.In one embodiment, the costimulatory domain comprises a protein selected from the group consisting of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating 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,CD8α, CD8β,IL2Rβ,IL2Rγ,IL7Rα,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,NKG2D,N KG2C,TNFR2,TRANCE / RANKL,DNAM1(CD226),SLAMF4(CD244,2B4),CD84,CD96(Tactile),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 a functional signaling domain of a protein that specifically binds to a ligand of CD83. In embodiments, the costimulatory domain comprises 4-1BB, CD27, CD28 or ICOS.

[0239] In one embodiment, the CAR molecule further comprises a sequence encoding an intracellular signaling domain, such as an intracellular signaling domain described herein. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 ζ. In one embodiment, the intracellular signaling domain comprises a sequence of SEQ ID NO: 16 and / or a sequence of SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises a sequence of SEQ ID NO: 16 and / or a 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 ζ. In one embodiment, the intracellular signaling domain comprises a sequence of SEQ ID NO: 51 and / or a sequence of SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises a sequence of SEQ ID NO: 51 and / or a sequence of SEQ ID NO: 43. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and the sequence of SEQ ID NO: 17 or SEQ ID NO: 43, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

[0240] In one embodiment, the CAR molecule further comprises a leader sequence, such as a leader sequence described herein. In one embodiment, the leader sequence comprises SEQ ID NO: 13 amino acid sequence or a sequence with SEQ ID NO: 13 amino acid sequence having 95-99% identity.

[0241] On the one hand, CAR (for example, CD19 CAR, ROR1 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, or CD79a CAR) include optional leader sequence (for example, optional leader sequence as described herein), extracellular antigen binding domain, hinge (for example hinge as described herein), membrane spaning domain (for example membrane spaning domain as described herein) and intracellular stimulation domain (for example intracellular stimulation domain as described herein).In one aspect, exemplary CAR constructs include optional leader sequence (for example, leader sequence as described herein), extracellular antigen binding domain, hinge, membrane spaning domain, intracellular costimulatory domain (for example intracellular costimulatory domain as described herein) and intracellular stimulation domain.

[0242] Bispecific antibodies

[0243] Bispecific antibody molecules (which can be, for example, administered alone or as part of a CAR) can include two VH regions and two VL regions. In some embodiments, upstream antibody or a portion thereof (e.g., scFv) is arranged so that its VH (VH1) is upstream of its VL (VL1), and downstream antibody or a portion thereof (e.g., scFv) is arranged so that its VL (VL2) is upstream of its VH (VH2), so that the entire bispecific antibody molecule has arrangement VH1-VL1-VL2-VH2. In other embodiments, upstream antibody or a portion thereof (e.g., scFv) is arranged so that its VL (VL1) is upstream of its VH (VH1), and downstream antibody or a portion thereof (e.g., scFv) is arranged so that its VH (VH2) is upstream of its VL (VL2), so that the entire bispecific antibody molecule has arrangement VL1-VH1-VH2-VL2.

[0244] Bispecific CD22 / CD19 inhibitors

[0245] 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 of Table 28 or a sequence with 95-99% identity thereto. Also provided is a nucleic acid according to Table 28 or a sequence with 95-99% identity thereto. In one embodiment, the B cell inhibitor comprises the CD19-specific antibody molecules of Table 2 or 3 (or a sequence with 95-99% identity thereto) and the CD22-specific antibody molecules of Table 6A or 6B (or a sequence with 95-99% identity thereto). In one embodiment, the B cell inhibitor comprises a CD19-specific antibody molecule with one or more CDRs of Table 4 or 5 (or with 1,2,3,4,5 or 6 changes, such as substituted sequences) and a CD22-specific antibody molecule with a CDR of Table 7A, 7B, 7C, 8A or 8B (or with 1,2,3,4,5 or 6 changes, such as substituted sequences).

[0246] MTOR inhibitors

[0247] In one embodiment, the cells of expression CAR molecules (such as CD19CAR molecules, CD20CAR molecules or CD22CAR molecules, such as CAR molecules described herein) optionally administered in combination with B cell inhibitors are co-administered with the mTOR inhibitors of low immune enhancing doses. Although it is not desired to be bound by theory, it is believed that treatment with low immune enhancing doses (for example, not enough to completely suppress the immune system but enough to improve immune function) is accompanied by a reduction 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 consumed by engaging with cells expressing PD-1 ligands such as PD-L1 or PD-L2.

[0248] In one embodiment, the method can be used to optimize the performance of CAR cells as described herein in a subject. Although it is not desired to be bound by theory, it is believed that in one embodiment, the performance of endogenous unmodified immune effector cells (such as T cells) is improved. Although it is not desired to be bound by theory, it is believed that in one embodiment, the performance of CAR expressing cells is improved. In other embodiments, it has been or will be engineered to express CAR cells, such as T cells can be treated in vitro by contacting with a certain amount of mTOR inhibitors, and the certain amount of mTOR inhibitors increase PD1 negative immune effector cells, such as the number of T cells or increase PD1 negative immune effector cells, such as T cells / PD1 positive immune effector cells, such as the ratio of T cells.

[0249] In one embodiment, before administering CAR expressing cells as described herein, such as T cells, it is started to administer the mTOR inhibitor of low immune enhancement dosage, such as allosteric inhibitors such as RAD001 or catalytic inhibitors. In one embodiment, CAR cells are administered after the mTOR inhibitor of sufficient time or sufficient dosage, so that the level of PD1 negative immune effector cells (such as T cells) or PD1 negative immune effector cells, such as T cells / PD1 positive immune effector cells, such as the ratio of T cells is at least temporarily increased.

[0250] In one embodiment, cells engineered to express a CAR, such as T cells, are harvested after a low, immune-enhancing dose of an mTOR inhibitor for a sufficient time or a sufficient dose such that the level of PD1-negative immune effector cells (e.g., T cells) in or harvested from the subject or the ratio of PD1-negative immune effector cells, such as T cells / PD1-positive immune effector cells, such as T cells, is at least temporarily increased.

[0251] Other features or embodiments of the compositions or methods described herein include one or more of the following:

[0252] In embodiments, the B cell inhibitor comprises an inhibitor of one or more of CD10, CD20, CD22, CD34, CD123, FLT-3 or ROR1. In embodiments, 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.

[0253] In embodiments, one or more cells expressing a CAR molecule that binds CD19 are administered simultaneously with, before, or after one or more B cell inhibitors.

[0254] In embodiments, the subject has or is identified as having a difference, eg, a statistically significant difference, between the determined levels compared to a reference level of one or more markers listed in Table 29 in the biological sample.

[0255] In embodiments, the subject has or is identified as having a difference between the determined signature compared to a reference signature in the signature of CD 19 in the biological sample (e.g., a mutation causing a frameshift or premature stop codon, or both).

[0256] In embodiments, the subject has, or is identified as having, a difference, eg, a statistically significant difference, between the determined levels compared to a reference level of Treg cells in the biological sample.

[0257] In one embodiment, the method comprises administering to the subject a therapeutically effective dose of a chimeric antigen receptor (CAR) therapy, e.g., a CAR therapy described herein, e.g., a therapy comprising CD19 CAR-expressing cells and optionally one or more B cell inhibitors, and the subject is identified as having a determined level compared to a reference level or a difference (e.g., a statistically significant difference) between a determined characteristic compared to a reference characteristic in one or more of the following: (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 results in a frameshift or premature stop codon or both, or (iii) a T cell marker in the biological sample. REG In one embodiment, the method comprises measuring (i) the level of one or more markers listed in Table 29; (ii) a characteristic of CD19, such as a mutation, such as a mutation that results in a frameshift or premature stop codon, or both; or (iii) the level of T cells in the biological sample. REG The method comprises determining whether the subject has a difference, such as a statistically significant difference, between the determined levels compared to the reference levels or between the determined characteristics compared to the reference characteristics in one or more of the levels or activities of the cells, and administering a therapeutically effective dose of a chimeric antigen receptor (CAR) therapy, such as a CAR therapy described herein, such as a therapy comprising CD19 CAR expressing cells and optionally one or more B cell inhibitors to the subject. In one embodiment, the method comprises determining (i) the level of one or more markers listed in Table 29; (ii) a characteristic of CD19, such as a mutation, such as a mutation that results in a frameshift or premature stop codon or both, or (iii) a T cell in the biological sample. REG The method comprises administering a therapeutically effective dose of a chimeric antigen receptor (CAR) therapy, such as a CAR therapy described herein, such as a therapy comprising CD19 CAR expressing cells and optionally one or more B cell inhibitors, to the subject, determining whether the subject has a difference between the determined levels compared to the reference levels or between the determined features compared to the reference features, such as a statistically significant difference, and administering a therapeutically effective dose of a chimeric antigen receptor (CAR) therapy, such as a CAR therapy described herein, such as a therapy comprising CD19 CAR expressing cells and optionally one or more B cell inhibitors. In one embodiment, the method comprises administering a therapeutically effective dose of a chimeric antigen receptor (CAR) therapy, such as a CAR therapy described herein, such as a therapy comprising CD19 CAR expressing cells, to the subject, determining whether the subject has (i) the level of one or more markers listed in Table 29; (ii) a feature of CD19, such as a mutation, such as a mutation that causes a frameshift or premature stop codon or both, or (iii) a T in a biological sample. REGThere is a difference, e.g., a statistically significant difference, in one or more of the level or activity of the cells between the determined levels as compared to the reference levels, or between the determined characteristics as compared to the reference characteristics, and if the difference exists, administering to the subject a therapeutically effective dose of one or more B cell inhibitors.

[0258] In embodiments, the subject has or is identified as having an increase, eg, a statistically significant increase, between the determined level and the reference level of Treg cells in the biological sample.

[0259] In embodiments, the subject has relapsed or is identified as having relapsed following treatment with one or more cells expressing a CAR molecule that binds CD19 (e.g., a CD19 CAR).

[0260] In embodiments, the B cell inhibitor comprises an effective number of one or more cells that express: a CAR molecule that binds to CD10, e.g., a CD10 CAR described herein; a CAR molecule that binds to CD20, e.g., a CD20 CAR described herein; a CAR molecule that binds to CD22, e.g., a CD22 CAR described herein; a CAR molecule that binds to CD34, e.g., a CD34 CAR described herein; a CAR molecule that binds to CD123, e.g., a CD123 CAR described herein; a CAR molecule that binds to FLT-3, e.g., a FLT-3 CAR described herein; or a CAR molecule that binds to ROR1, e.g., a ROR1 CAR described herein.

[0261] In embodiments, the CD19 inhibitor comprises an antibody or antibody fragment comprising a CD19 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, and wherein the CD19 binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of any CD19 light chain binding domain amino acid sequence listed in Table 2 or Table 3, and one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of any CD19 heavy chain binding domain amino acid sequence listed in Table 2 or Table 3.

[0262] In embodiments, the CD19 CAR comprises a light chain variable region listed in Table 2 or 3 and any heavy chain variable region listed in Table 2 or 3.

[0263] In embodiments, the CD19 inhibitor comprises a CD19 binding domain comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, or a sequence with 95-99% identity thereof. In embodiments, the CD19 CAR comprises a polypeptide of SEQ ID NO: 58.

[0264] In embodiments, the B cell inhibitor comprises a CD20 CAR comprising an antibody or antibody fragment comprising a CD20 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, and wherein the CD20 binding domain comprises one or more light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of any CD20 light chain binding domain amino acid sequence listed in Table 13, and one or more heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of any CD19 heavy chain binding domain amino acid sequence listed in Table 12A or 12B.

[0265] In embodiments, the B cell inhibitor comprises a CD22 CAR comprising an antibody or antibody fragment comprising a CD22 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, and wherein the CD22 binding domain comprises one or more light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 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 complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of any CD22 heavy chain binding domain amino acid sequence listed in Tables 7A, 7B, 7C, 9A, and / or 9B.

[0266] In embodiments, the CD22 CAR comprises any of the light chain variable regions listed in Table 10A or 10B. In embodiments, the CD22 CAR comprises any of the heavy chain variable regions listed in Table 9A or 9B. In embodiments, the CD22 CAR comprises 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.

[0267] In embodiments, the B cell inhibitor comprises a CAR comprising an antibody or antibody fragment comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, and wherein the antigen binding domain comprises one or more (e.g., all) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3), and one or more (e.g., all) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3).

[0268] In embodiments, the B cell inhibitor comprises a CAR comprising an scFv. In embodiments, the B cell inhibitor comprises a CAR comprising a transmembrane domain comprising an α, β or ζ chain selected from the group consisting of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In embodiments, the antigen binding domain is connected to the transmembrane domain via a hinge region. In embodiments, the hinge region comprises SEQ ID NO: 14 or a sequence thereof with 95-99% identity. In embodiments, the costimulatory domain is a functional signaling domain obtained from a protein selected from OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).In an embodiment, the costimulatory domain is selected from the group consisting of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating 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,CD8α,C D8β,IL2Rβ,IL2Rγ,IL7Rα,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,NKG2D,NKG 2C,TNFR2,TRANCE / RANKL,DNAM1(CD226),SLAMF4(CD244,2B4),CD84,CD96(Tactile),CEACAM1,CRTAM,Ly9(C In some embodiments, the costimulatory domain comprises a functional signaling domain obtained from a protein that specifically binds to a ligand of SEQ ID NO: D229), 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 a ligand that specifically binds to CD83. In some embodiments, the costimulatory domain comprises a sequence of SEQ ID NO: 16 or SEQ ID NO: 51. In some embodiments, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3ζ.

[0269] In embodiments, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and / or the sequence of SEQ ID NO: 17 or SEQ ID NO: 43. In embodiments, the CAR further comprises a leader sequence. In embodiments, the leader sequence comprises SEQ ID NO: 13.

[0270] In embodiments, the cell expressing the CAR molecule comprises a T cell or a NK cell.

[0271] In embodiments, the disease associated with CD19 expression is selected from a proliferative disease such as cancer or malignancy or a precancerous condition such as myelodysplasia, myelodysplastic syndrome or preleukemia, or a non-cancer related indication associated with CD19 expression. In embodiments, the disease is a 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) or chronic lymphocytic leukemia (CLL) One or more.

[0272] In embodiments, the method further comprises administering an agent that increases the efficacy of cells expressing CAR molecules. In embodiments, the method further comprises administering an agent that improves one or more side effects associated with administering cells expressing CAR molecules. In embodiments, cells expressing CAR molecules are administered in combination with an agent for treating a disease associated with CD19.

[0273] In embodiments, according to the methods described herein, for example, methods of providing anti-tumor immunity to a mammal or methods of treating a mammal, the mammal is a non-responder, partial responder, or complete responder to a previously administered cancer therapy, such as a CD19 CAR therapy or a cancer therapy other than CD19 CAR expressing cells. In embodiments, the mammal is a non-relapser, partial relapser, or complete relapser of a previously administered cancer therapy, such as a CD19 CAR therapy or a cancer therapy other than CD19 CAR expressing cells. In embodiments, the mammal comprises CD19 negative cancer cells or CD19 positive cancer cells, optionally wherein the mammal further comprises 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 is pre-administered with CD19 CAR expressing cells and is refractory to CD19 CAR treatment.

[0274] In embodiments, the active agent is an mTOR inhibitor and the subject is administered a low, immune-enhancing dose of an mTOR inhibitor, such as RAD001 or rapamycin. In embodiments, the mTOR inhibitor is RAD001. In embodiments, the dose comprises an allosteric and catalytic mTOR inhibitor. In embodiments, the mTOR inhibitor is administered for an amount of time sufficient to reduce the proportion of PD-1 positive T cells in the subject's peripheral blood or a preparation of T cells isolated from the subject, increase the proportion of PD-1 negative T cells, or increase the ratio of PD-1 negative T cells / PD-1 positive T cells.

[0275] In embodiments, to be engineered to express CAR immune effector cells (e.g., T cells) after a sufficient time or after a low immune enhancement dose of an mTOR inhibitor is administered in sufficient amounts, it is harvested so that the PD1 negative immune effector cells, such as the level of T cells, or the ratio of PD1 negative immune effector cells (e.g., T cells) / PD1 positive immune effector cells (e.g., T cells) in the subject are harvested. In embodiments, the dosage of the mTOR inhibitor is associated with at least 5 but no more than 90% mTOR inhibition, for example, by p70 S6 K inhibition measurement. In embodiments, the dosage of the mTOR inhibitor is associated with at least 10% but no more than 40% mTOR inhibition, for example, by p70 S6 K inhibition measurement.

[0276] In one embodiment, the method further comprises administering a checkpoint inhibitor. In embodiments, the subject receives pre-treatment with an active agent such as an mTOR inhibitor and / or a checkpoint inhibitor before starting CART therapy. In embodiments, the subject receives simultaneous treatment with an active agent such as an mTOR inhibitor and / or a checkpoint inhibitor. In embodiments, the subject receives treatment with an active agent such as an mTOR inhibitor and / or a checkpoint inhibitor after CART therapy.

[0277] In an embodiment, the determined level or determined characteristic is obtained before, concurrently with, or during CART therapy.

[0278] In embodiments, the method includes determining whether the gene signature indicating whether the subject may relapse or has relapsed. In embodiments, the method includes determining the gene signature in the subject before using CAR-expressing cell therapy, such as CART therapy (e.g., CART19 therapy, such as CTL019 therapy), which predicts the recurrence of CAR therapy. In embodiments, the level of one or more markers is the level of at least 2,3,4,5,6,7,8,9 or 10 markers listed in Table 29. In embodiments, the level of marker includes the level of mRNA level or soluble protein.

[0279] In embodiments, CD19 is characterized by a mutation in exon 2, such as a mutation that causes a frameshift or a premature stop codon, or both. In embodiments, the expression of T REG Stain samples for markers expressing cells to determine T REG In one embodiment, T REG The level of cells is the level of Treg cells in a relevant location within the subject's body, such as within a cancer microenvironment.

[0280] In embodiments, the method further comprises reducing T in the subject prior to apheresis. REG In embodiments, the method further comprises reducing T in the subject, for example by administering cyclophosphamide, an anti-GITR antibody, or both to the subject. REG Features. In embodiments, the method includes pre-treating the subject with cyclophosphamide, anti-GITR antibody, or both before collecting cells for CAR-expressing cell product manufacturing. In embodiments, the method further includes obtaining a sample from the subject, wherein the sample comprises a cell fraction (e.g., it comprises blood), a tissue fraction, an apheresis sample, or a bone marrow sample.

[0281] In embodiments, the cell expresses an inhibitory molecule comprising a first polypeptide comprising at least a portion of an inhibitory molecule bound to a second polypeptide comprising a positive signal from an intracellular signaling domain. In embodiments, the inhibitory molecule comprises a first polypeptide comprising at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and a primary signaling domain.

[0282] In embodiments, the method comprises determining a gene signature that indicates whether a cell-treated subject is likely to relapse or has relapsed. In embodiments, the method comprises determining a gene signature in the cells prior to infusion into the subject. In embodiments, the method further comprises reducing T in a cell population comprising the transduced cells. REG In an embodiment, reducing T REG Characterization includes CD25 depletion of the cell population.

[0283] In embodiments, the subject is a mammal, such as a human.

[0284] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references (e.g., sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene and Entrez sequences cited herein (e.g., in any table herein) are incorporated by reference herein. Unless otherwise indicated, sequence accession numbers specified herein (including in any table herein) refer to database entries current as of April 8, 2015. When a gene or protein references multiple sequence accession numbers, all sequence variants are included.

[0285] In addition, the materials, methods, and examples are illustrative only and not limiting.

[0286] Headings, subheadings, or numbered or lettered elements, such as (a), (b), (i), etc., are presented solely for ease of reading. The use of headings or numbered or lettered elements in this document does not require that the steps or elements be performed in alphabetical order or that the steps or elements be discrete from one another.

[0287] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0289] Figure 1A and 1B is a schematic diagram of a representative CAR.

[0290] Figure 2 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.

[0291] Figure 3 Schematic diagram of the experimental setup for the study evaluating the therapeutic efficacy of CART19 therapy in patients with Hodgkin lymphoma.

[0292] Figure 4A , 4B, 4C, and 4D show flow cytometric analysis of PD1 and CAR19 expression on T cells. Figure 4A and 4B Representative flow cytometry profiles demonstrating the distribution of PD-1 and CAR19 expression on CD4+ T cells from subjects who were complete responders (CR) or non-responders (NR) to CART therapy. Figure 4Cis a graph showing the percentage of PD1 cells in CD4+ T cell populations from groups of subjects with different responses to CART therapy. Figure 4D is a graph showing the percentage of PD1 cells in the CD8+ T cell population for subject groups with different responses to CART therapy.

[0293] Figure 5A and 5B CD4 and CAR19 expressing cells from groups of subjects with different responses to CART therapy are shown ( Figure 5A ) or CD8 and CAR19 expressing cells ( Figure 5B ) Distribution of PD1 expression in .

[0294] Figure 6 Shown are flow cytometric analyses of PD1, CAR19, LAG3, and TIM3 expression on T cells from subjects who were complete responders (CR) or non-responders (NR) to CART therapy.

[0295] Figure 7A and 7B Showing PD1 and LAG3 expression from groups of subjects with different responses to CART therapy ( Figure 7A ) or PD1 and TIM3 expression ( Figure 7B ) distribution.

[0296] Figure 8 shows plasma cell IgA immunophenotyping analysis of myeloma patients who received CART19, confirming response to CART19 therapy.

[0297] Figure 9A and 9B Figure 1 shows the expression of IL-7 receptor (CD127) on cancer cell lines and CART cells. The expression of CD127 was determined by flow cytometry analysis in three cancer cell lines: RL (mantle cell lymphoma), JEKO (also known as Jeko-1, mantle cell lymphoma) and Nalm-6 (B-ALL). Figure 9A CD127 expression on infused and circulating CD3-positive (CART) cells in NSG mice was determined by flow cytometric analysis ( Figure 9B ).

[0298] Figure 10A , 10B and 10C show the anti-tumor response after CART19 treatment and subsequent IL-7 treatment. NSG mice transplanted with a luciferase-expressing mantle cell lymphoma cell line (RL-luc) on day 0 were treated with different doses of CART19 cells on day 6, and tumor burden was monitored. The mice were divided into 4 groups, receiving no CART19 cells, 0.5x10 6CART19 cells (CART19 0.5E6), receiving 1x10 6 CART19 cells (CART19 1E6) or 2x10 6 CART19 cells (CART19 2E6). Tumor burden after CART treatment was measured by detecting bioluminescence (average BLI) ( Figure 10A ). will accept 0.5x10 6 CART19 cells (CART19 0.5E6) or 1x10 6 Mice that were injected with CART19 cells (CART19 1E6) were randomly assigned to receive or not receive recombinant human IL-7 (rhIL-7). Figure 10A Tumor burden as indicated by mean bioluminescence (BLI) was monitored in three mice (#3827, #3829, and #3815, receiving the indicated initial CART19 doses) treated with IL-7 starting on day 85 ( Figure 10B IL-7 was administered 3 times a week by IP injection. Tumor burden expressed by mean bioluminescence (BLI) before day 85 (PRE) and after day 115 (POST) was compared between mice not receiving IL-7 (CTRL) and mice treated with IL-7 (IL-7) ( Figure 10C ).

[0299] Figure 11A and 11B T cell dynamics after IL-7 treatment are shown. The levels of human T cells detected in the blood were monitored for each mouse that received IL-7 or control mice ( Figure 11A The 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 ).

[0300] Figure 12 Depicted are the structures of two exemplary RCAR configurations. Antigen binding members include antigen binding domains, transmembrane domains, and switch domains. Intracellular binding members include switch domains, costimulatory signaling domains, and primary signaling domains. These two configurations demonstrate that the first and second switch domains described herein can be in different orientations relative to antigen binding members and intracellular binding members. Other RCAR configurations are further described herein.

[0301] Figure 13 Two constructs of bispecific CARs with anti-C22 and anti-CD19 binding domains are described."4G4S" indicates the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 1311).

[0302] Figure 14 is a graph depicting the activity of bispecific CD19 / CD22 CAR constructs in the NFAT assay.

[0303] Figure 15A , 15B, 15C are graphs showing the degree of CART cell activation (measured by relative luminescence) in the presence of various tumor target cell lines. Figure 15A Shown is CAR T cell activation in the presence of the CD20-expressing target cell line Daudi. Figure 15B Shown is CAR T cell activation in the presence of the CD20-expressing target cell line Raji. Figure 15C Shown is CAR T cell activation in the presence of the non-CD20 expressing negative control K562.

[0304] Figure 16 Figure 2 is an exemplary schematic diagram illustrating the overview of gene signature analysis. Briefly, for each gene set, two sets of statistical models were applied to determine whether the metagenes between CRs, PRs, and NRs were statistically different. CRs were more likely to be resting T EFF cells, while NR is more like an activated T EFF In activated and resting T cells EFF Genes upregulated in cells were also upregulated in NRs.

[0305] Figure 17 Depicted are exemplary results (p=0.000215) showing that T in samples from pediatric patients who were complete responders (R) who became relapsers compared to complete responders (CR) who did not relapse. REG The x-axis is the samples of the response group, where CR = complete responder without relapse, R = relapser. The y-axis is the normalized metagene expression score.

[0306] Figure 18A , 18B and 18C are figures showing CAR T cell activation in the presence of tumor target cell lines. Figure 18A CAR-expressing JNL cells were mixed with Daudi CD22-expressing target cell lines at the indicated E:T ratios. Figure 18B CAR-expressing JNL cells were mixed with Raji CD22-expressing target cell line at the indicated E:T ratios. Figure 18C CAR-expressing JNL cells were mixed with the negative control K562 cell line at the indicated E:T ratios.

[0307] Figure 19 is a graph showing primary T cell expression of chimeric antigen receptors on the cell surface. Figure 19-1 and Figure 19-2) and rhCD22-Fc / anti-Fc488 ( Figure 19-3 and 19-4 ) was used to determine the level of CAR surface expression. Cells without CAR were used as negative controls.

[0308] Figure 20A , 20B, 20C, 20D, 20E and 20F are figures showing primary T cell tumor target killing assays. Primary T cells activated and transduced with CD22CAR were mixed with target cell lines stably expressing luciferase at the indicated ratios, and target cell killing was measured. The percentage of killing was normalized to hCD22-8 (28.8% transduction). Compared with the positive control CD22 CAR m971 (m971-HL), the negative control CAR m971-LH and the untransduced T cells as a negative control, the CD22-expressing cell line Raji ( Figure 20A ), SEM( Figure 20B ), K562-hCD22( Figure 20C ), Daudi( Figure 20D ) and Nalm6( Figure 20E ) to test functional CD22 CAR clones. K562 cell line does not express CD22 and was used as a negative control ( Figure 20F ).

[0309] Figure 21A , 21B, 21C, 21D, 21E and 21F are graphs showing significant proinflammatory cytokine responses induced by CD22 CAR clones. Primary T cell killing assays were used to determine the ability of CD22 CAR clones to produce proinflammatory cytokines IFN-g, IL-2 and TNFa. Effector cells were co-cultured with each different target cell line normalized to 28.8% transduction for 20 hours. Supernatants were taken from different cultures and from target cells expressing Raji CD22 ( Figure 21A ), target cells expressing Nalm6 CD22 ( Figure 21B ), target cells expressing Daudi CD22 ( Figure 21C ), target cells expressing SEM CD22 ( Figure 21D ), target cells expressing K562-hCD22CD22 ( Figure 21E ), and K562 non-CD22 expressing cells (negative control) ( Figure 21F ) with different E:T ratios of 2.5:1 and 10:1.

[0310] Figure 22Figure 2 is a graph depicting the expression of various B cell antigens in relapsed ALL as detected by flow cytometry. Samples from 16 r / r patients were screened by multiparameter flow cytometry for the following markers: CD19 (16 pts), CD22 (16 pts), CD123 (16 pts), FLT-3 (9 pts), ROR-1 (3 pts), CD79b (15 pts), CD179b (8 pts), CD79a (16 pts), CD10 (16 pts), CD34 (16 pts), and CD20 (16 pts). CD22 and CD123 are highly expressed (>60%) and homogenously in the blasts of r / r ALL patients (the bars represent the median % expression, 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.

[0311] Figure 23 This is a set of graphs showing the expression of CD22 and CD123 in 6 patients with relapsed CD19-negative leukemia before (baseline) and after (CD19-negative relapse) CART19 treatment. In all analyses, the population of interest was gated based on forward and side scatter characteristics, then single-peak gated, and live cells were gated using Live Dead Aqua (Invitrogen). Time gating was incorporated into quality control. Gating strategies included: time gating → SSC low → single peak → live → CD45dim → CD10+.

[0312] Figure 24 It is a set of figures showing the expression of CD22 in blasts of patients with CD19-negative disease relapse after CART19 treatment (clinical trial UPCC04409 / CHP959, with patient UPN indicated in the box). The top row shows CD19 and CD22 expression in blasts before CART19 treatment, while the bottom row shows the disease phenotype at relapse. When CD19 expression is lost, CD22 expression is also maintained at relapse.

[0313] Figure 25 Is a set of graphs showing the expression of CD123 in blasts of patients with CD19-negative disease relapse after CART19 treatment (clinical trial UPCC04409 / CHP959, patient UPN is indicated in the box). The top row shows CD19 and CD123 expression in blasts before CART19 treatment, while the bottom row shows the disease phenotype at relapse. When CD19 expression is lost, CD123 expression is maintained in most patients at relapse.

[0314] Figure 26 Figure 2 is a graph showing the median expression of CD19, CD22, and CD123 before and after CART19 treatment in patients with CD19-negative disease relapse. 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) were still expressed. For each patient, the percentage of cells expressing the marker is shown as a single data point.

[0315] Figure 27A and 27B is a series of graphs showing CD22 expression in samples from 16 r / r ALL patients and 4 patients with relapsed CD19-negative disease after treatment with CART19 therapy. Samples were screened for the B cell marker CD22 by multiparameter flow cytometry. CD22 was expressed at high levels (>60%) and homogenously in the blasts of 11 / 15 r / r ALL patients ( Figure 27A In 4 / 4 patients with relapsed CD19-negative leukemia before (baseline) and after (CD19-negative relapse) CART19 treatment (shown as 2pts), CD22 was positive ( Figure 27B ). Gating strategy: SSC low → singlet → live → CD45 dim.

[0316] Figure 28A , 28B and 28C are a series of figures showing the effect of CD22 CART on CD19 and CD22 expression. A scheme showing two CAR22 constructs generated using different chain orientations (H to L and L to H) is shown ( Figure 28A The anti-CD22 scFv (m971) was codon-optimized and cloned into a murine CAR19 vector containing the CD8 hinge, 41-BB costimulatory and CD3ζ signaling domains ( Figure 28A The expression of CD19, CD22 and isotype control on NALM6 ALL cell line is shown as mean fluorescence intensity (MFI) ( Figure 28B ) and antibody binding capacity (ABC)( Figure 28C In NALM-6, CD19 expression is higher than CD22. However, in most primary ALL samples, CD19 and CD22 expression are similar (see Figure 27A ).

[0317] Figure 29A, 29B and 29C are a series of graphs showing the expansion of normal donor T cells used to generate CART22 and CART19 (along with UTD cells). Population doublings (PD) versus culture days: At the end of expansion (day 11), CART22 and control T cells reached approximately 4.5 PD, which was not significantly different from CART19 or UTD cells ( Figure 29A ). T cell volume (fl) versus culture days: There was a peak volume (approximately 450 μl) on day 6, while the volume decreased to 300 fl in subsequent days when the cells were harvested and frozen. No significant differences were observed with CART19 or UTD cells ( Figure 29B CAR expression on CD4-positive and CD8-positive T cells on day 11 of expansion is shown in Figure 2. Figure 29C As shown. Gating for CAR expression was based on UTD. Gating strategy: FSS vs. SSC lymphocytes → singlets → viable → CD3+.

[0318] Figure 30 Figure 2 is a series of graphs showing CD107a degranulation assays with cytoplasmic cytokine production. CART19, CART22HtoL and LtoH were co-cultured with different targets (alone, PMA / IONOMYCIN, MOLM-14 and NALM-6). When co-cultured with an ALL cell line (NALM-6) but not with a negative control, CART19 and CART22HtoL showed high levels of CD107a degranulation, IL-2, IFNg and TNFa production. UTD and CART22 LtoH did not show degranulation or cytokine production. Gating strategy: FSS vs. SSC lymphocytes → singlet → live → CD3+.

[0319] Figure 31 This figure shows a luciferase-based killing assay. CART22 and CART19 HtoL, but not UTD cells, were able to lyse NALM-6 cells after 24 hours of co-culture. A direct correlation between cytotoxic activity and the E:T ratio was observed, with a 2:1 E:T ratio exhibiting superior anti-leukemic efficacy (78% and 75% killing for CART19 and CART22, respectively).

[0320] Figure 32A and 32B Is a series of graphs showing CFSE-based proliferation assays. CART22 and CART19 co-cultured with the ALL cell line NALM-6 for 5 days resulted in significant T cell proliferation (94% and 92.9%, respectively). Controls (TCM = culture medium alone, PI = PMA / ionomycin, MOLM-14) ( Figure 32AIn the bar graph showing the kinetics of CFSE dilution in CART19 and CART22, most T cells undergo multiple cycles of proliferation ( Figure 32B ). Gating strategy: FSS vs. SSC lymphocytes → singlets → live → CD3+.

[0321] Figure 33 It is a series of graphs showing cytokine production. CART22, CART19 and UTD were incubated with different radiation targets (alone, PMA / ionomycin, MOLM-14 and NALM-6) for 24 hours. When co-cultured with the ALL cell line NALM-6, only CART22 and CART19HtoL were able to release multiple cytokines (IFNg, IL-2, GM-CSF, TNFa and MIP1b shown here). The results are shown as mean fluorescence intensity (MFI).

[0322] Figure 34A and 34B is a series of graphs showing T cell degranulation with primary ALL blasts. CART22, CART19, and UTD cells were co-incubated for 4 hours with blasts from an ALL patient (CHP-959-101) at baseline and after CART19 treatment when the patient relapsed with CD19-negative disease. Both CART19 and CART22 were able to degranulate at baseline (when the blasts were CD19+ and CD22+), but at relapse, only CART22 degranulated (when the disease was CD19-neg) ( Figure 34A The dot plot of CD107a degranulation in CD8-pos and CD8-neg CART19 and CART22 effectors following incubation with CHP101 samples at relapse showed that only CART22 showed degranulation in both CD8 and CD4 T cells ( Figure 34B ). Gating strategy: FSS vs. SSC lymphocytes → singlets → live → CD3+.

[0323] Figure 35A , 35B, 35C and 35D are a series of figures showing the in vivo CART22 efficacy against NALM-6. A. Experimental protocol: 1 million NALM-6 luciferase+ cells / mouse were injected intravenously into NSG mice. After 6 days, tumor implantation was assessed by bioluminescence. Mice were then randomly assigned to receive untransduced T cells or different doses of CART22 (1.25 to 5 million total cells / mouse with 75% CAR expression). The mice were then monitored for tumor burden, PB T cell expansion and survival ( Figure 35A ). Dose-related anti-leukemic responses were detected by bioluminescence (BLI) tumor burden. 6 Mice with CART22 cells showed better tumor control ( Figure 35B). Mice treated with CART22 showed statistically significant better overall survival (OS) compared to mice treated with UTD cells. For OS, there was a significant correlation between higher doses of CART22 and better OS ( Figure 35C ). T cell expansion in vivo was monitored weekly by retroorbital bleeding. One week after T cell infusion, mice receiving a higher dose of CART22 showed better CART expansion (median of 12 T cells / μl) ( Figure 35D ).

[0324] Figure 36A and 36B Is a series of figures showing the in vivo comparison between CART22 and CART19 against NALM-6. Experimental protocol: 1 million NALM-6 luciferase+ cells / mouse were injected intravenously in NSG mice. After 6 days, tumor implantation was assessed by bioluminescence. Mice were then randomly assigned to receive untransduced T cells, CART19 or CART22 (5 million total cells, 75% CAR expression). The mice were then monitored for tumor burden, PB T cell expansion and survival ( Figure 36A Tumor burden, as measured by bioluminescence (BLI), demonstrated an anti-leukemic response in CART22- and CART19-treated mice, whereas UTD mice rapidly progressed ( Figure 36B ). CART19-treated mice showed better overall survival (OS) compared with CART22, possibly due to different target expression in NALM-6 (CD19>>CD22)( Figure 36C ).

[0325] Figure 37A and 37B is a series of figures showing an in vivo comparison between CART22 and CART19 in a model of primary ALL. Embryonic cells from a primary ALL patient (JH331) were passaged in vivo and transduced with luciferase to track tumor burden. Experimental protocol: 1 million JH331 luciferase+ cells / mouse were injected intravenously into NSG mice. After 14 days, tumor implantation was assessed by bioluminescence. Mice were then randomized to receive untransduced T cells, CART19 or CART22 (5 million total cells, 75% CAR expression). The mice were then monitored for tumor burden, PBT cell expansion and survival ( Figure 37A Tumor burden monitored by bioluminescence (BLI) detected an anti-leukemic response in CART22- and CART19-treated mice, whereas UTD mice rapidly progressed ( Figure 37B ).

[0326] Figure 38A, 38B and 38C are a series of images of tissue microarrays showing CD22 expression on 28 normal human tissues by immunohistochemical staining. Lymphoid organs were positive for CD22 expression (tonsils, lymph nodes, spleen and thymus) ( Figure 38A Non-lymphoid organs did not show expression of CD22 ( Figure 38B CD22-positive resident B cells were observed in multiple tissues ( Figure 38C ). * = nonspecific staining.

[0327] 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 lymphoblastoid cells and leukemia / lymphoma cell lines were also highly positive.

[0328] Figure 40 Figure 2 is a series of graphs showing 51-chromium release assays of CART22 toxicity. Both CART22 and CART19, but not UTD cells, triggered lysis of the ALL cell line NALM-6. No cytotoxic effects of CART22 were observed in any normal tissue (CD34+, human neuronal progenitor cells or neurons and keratinocytes) or control (K562 cell line).

[0329] Figure 41 A graphical representation of CAR expression in JNL cells transduced with anti-CD123 CAR constructs assessed by FACS is shown 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.

[0330] Figure 42A , 42B and 42C show diagrams of CD123 CAR activity in JNL cells. The activity of anti-CD123 CAR constructs was assessed using a Jurkat cell line containing a luciferase reporter gene driven by an NFAT promoter (referred to as JNL cells). CAR activity was measured as the activation of this NFAT-driven reporter gene.

[0331] Figure 43A and 43B CD123 expression and activity are shown. Figure 43AShown is a graphic representation of CD123 CAR expression in primary T cells. Using protein L as a detection reagent, the percentage of transduced cells (expressing anti-CD123 CAR on the cell surface) and their relative fluorescence intensity were determined by flow cytometry analysis on BD LSRFortessa or BD-FACSCano. The gated histogram of the relative fluorescence intensity of the signal higher than that of unstained cells from the FACS shows the percentage of transduced T cells. Transduction results in a CAR positive cell range of 12-42%. Figure 43B Figure 1 shows a graphic representation of CD123-CART-mediated cell killing. T cell killing is directed against MOLM13 acute myeloid leukemia cells expressing CD123 that stably express luciferase. Untransduced T cells were used to determine the level of nonspecific background killing. The cytolytic activity of CART-CD123 was measured in an effector cell: target cell ratio of 4: 1 and a T cell dilution of 2 times, wherein effector cells are defined as T cells expressing anti-CD123 chimeric receptors. The assay was initiated by mixing an appropriate number of T cells with a constant number of target cells. After 20 hours, the cells were analyzed using Bright-Glo on an EnVision instrument. TM Luciferase assay measures luciferase signal.

[0332] Figure 44A and 44B The transduction efficiency of T cells using CD123-CAR is shown. Figure 44A The transduction efficiency of T cells using 1172 and 1176 is shown. Figure 44B Shown are the transduction efficiencies of T cells using CD123 CARs 2-4.

[0333] Figure 45 Flow cytometry of CD123 CAR2-4 and 1172 and 1176 is shown to determine the CD4:CD8 ratio.

[0334] Figure 46 shows the degranulation of CD123 CAR2-4, 1172, and 1176 upon exposure to CD123+ tumor cells.

[0335] Figure 47 Shown is a graphic representation of a luciferase assay used to evaluate the cytotoxicity of CART cells (NVS2-4, 1172 and 1176 clones) against tumor target cells (MOLM14).

[0336] Figure 48 Shown is a comparison of tumor burden in NSG mice injected with luciferase-expressing MOLM14 cells at D6 (before CART injection) and on day 13 (6 days after injection of NVS2-4, 1172, or 1176 clones) or day 20.

[0337] Figure 49A , 49B, 49C, 49D, 49E, and 49F show that CD123 is highly expressed in CD19-neg B-cell acute lymphoblastic leukemia relapse that occurs after CART19 treatment. Figure 49A Shown is the expression of CD123 compared with CD19 in 42 relapsed / refractory ALL samples. Figure 49B Co-expression of CD123 and CD19 in B-ALL blasts is shown. Gating was performed on blasts (SSC low, singlet, viable, CD45 dim). Figure 49C The gating strategy for leukemic stem cells (LSCs) is shown. CD123 is highly expressed in this subset. Figure 49D Shown are the results of CD123 and CD19 co-expression and FISH analysis. Figure 49E and 49F Shown is the comparison of CD19 and CD123 expression at baseline or after relapse.

[0338] Figure 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 showed CD19 and CD123 expression; Figure 50B CD107a degranulation assay is shown; Figure 50C demonstrated the ability to target cell killing; Figure 50D and 50E Shows proliferation ability; Figure 50F Cytokine production is shown for the indicated cytokines.

[0339] Figure 51A , 51B and 51C showed that CART cells expressing CD19 CAR (CAR19) or CD123 CAR (CAR123) had anti-tumor effects in an in vivo mouse model. Figure 51A Tumor burden represented by bioluminescence imaging was shown; Figure 51B Shows the overall survival curve of mice receiving CART therapy; and Figure 51C Showing the expansion of CART123 cells in peripheral blood.

[0340] Figure 52A , 52B, 52C, 52D, 52E and 52F showed that CART123 was active in an in vivo mouse model of antigen-deficient relapse. Figure 52A The experimental scheme is shown; Figure 52B Disease progression by bioluminescence imaging at baseline and relapsed disease relative to CD19 expression (top panel) and in response to CART19 therapy treatment (bottom panel) is shown. Figure 52CBioluminescence images of mice administered with untransduced T cells or CART19 cells are shown. Figure 52D Show experimental protocols for treatment with CART19 or CART123; Figure 52E Shows disease progression; and Figure 52F Overall survival of treated mice is shown.

[0341] Figure 53A , 53B and 53C show ALL-CART interactions in the calvarial bone marrow of xenografted mice. Figure 53A The experimental scheme is shown; Figure 53B Representative multiphoton XY plane images of CART19 cells and CART123 cells interacting with ALL tumors engineered to express CD19 and CD123 or CD123 alone are shown (active cells are represented by dashed circles and inactive cells are represented by arrows); and Figure 53C is a graphical representation of a microscope image.

[0342] Figure 54A , 54B and 54C showed the prevention of CD19-neg relapse using CART19 and CART123. Figure 54A The experimental scheme is shown; Figure 54B Shown are disease progression (tumor burden represented by BLI) in mice treated with untransduced T cells (top panel), CART19 (middle panel), or a combination of CART19 and CART123 (bottom panel); and Figure 54C Overall survival is shown for this experiment.

[0343] Figure 55A and 55B Showing expression of CAR19 and CAR123 ( Figure 55A ) of T cells and degranulation assay results ( Figure 55B ).

[0344] Figure 56A and 56B Features of ALL blasts are shown. Figure 56A Shows expression of various markers CD19, CD123, CD10, CD34, and CD20; and Figure 56B The gating strategy for sorting CD19-CD123+ cells is shown.

[0345] Figure 57A , 57B, 57C, and 57D showed the anti-leukemia activity of CART123. Figure 57A The expression of CD19 and CD123 on NALM6 cells is shown; Figure 57B Tumor burden (indicated by BLI) in response to CART19 or CART123 therapy is shown; Figure 57CShow overall survival of mice administered CART19 or CART123; and Figure 57D Shown are the overall survival times of mice administered different doses of CART123.

[0346] Figure 58A and 58B Characterization of an in vivo model of antigen loss relapse is shown. Figure 58A represents the expression of CD123 in CD123-negative relapsed disease; and Figure 58B Degranulation assays of CART19 or CART123 cells when cultured in vitro with baseline or relapse cells are shown.

[0347] Figure 59 The results show that low doses of RAD001 enhance the proliferation of transduced T cells expressing CAR in a cell culture system. CART was 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 (white) was assessed after 4 days of co-culture.

[0348] Figure 60 Figure 3. Tumor growth measurements of NALM6-luc cells following daily RAD001 dosing at 0.3, 1, 3, and 10 mg / kg (mpk) or vehicle administration. Circles represent vehicle; 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 diamonds represent 0.3 mg / kg dose of RAD001.

[0349] Figure 61A and 61B Shown are pharmacokinetic curves showing the amount of RAD001 in the blood of NSG mice bearing NALM6 tumors. Figure 61A Shown is the day 0 PK of RAD001 after the first dose. Figure 61B Shown are the PK data after the final RAD001 dose on day 14. Diamonds represent the 10 mg / kg dose of RAD001; squares represent the 1 mg / kg dose of RAD001; triangles represent the 3 mg / kg dose of RAD001; and x represents the 10 mg / kg dose of RAD001.

[0350] Figure 62A and 62B The in vivo proliferation of humanized CD19 CART cells with and without RAD001 administration was shown. Low doses of RAD001 (0.003 mg / kg) per day resulted in enhanced CAR T cell proliferation, exceeding the normal level of huCAR19 proliferation. Figure 62A CD4+CAR T cells were shown; Figure 62B CD8+ CAR T cells are shown. 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 circles represent huCTL019 with 0.003 mg / kg RAD001.

[0351] Figure 63 Shown are multiplexed FIHC AQUA analyses demonstrating significant differences between CD3+ / PD-1+ cell populations in primary and secondary human DLBCL patient samples.

[0352] Figure 64 AQUA analysis showing various levels of CD19 (lower panel) and PD-L1 (upper panel) in primary and secondary sites of DLBCL samples. 40 human DLBCL patient samples, 25 primary and 15 secondary sites, were subjected to multiplex FIHC followed by AQUA analysis to identify the expression levels of CD19 and PD-L1 proteins.

[0353] Figure 65 Shown is a schematic diagram of two populations of CAR-expressing cells. In the population on the left (merged), each cell expresses one type of CAR. In the population on the right (dual cis CAR), each cell expresses two types of CAR.

[0354] Figure 66 Diagrams of dual cis CARs are shown. The upper CAR has a CD19 CAR and a CD22 CAR separated by a P2A protease cleavage site. The lower CAR has a CD19 CAR and a CD123 CAR separated by a P2A protease cleavage site.

[0355] Figure 67 Co-expression of CD19 and CD22 CARs from a bicistronic vector is shown.

[0356] Figure 68 , Top panel shows co-expression of CD19 and CD123 CARs from a bicistronic vector. Figure 68 , bottom panel, shows the anti-leukemic effect of these cells.

[0357] Figure 69 Shown are tumor burdens in mice bearing CD19-negative B-ALL xenografts after treatment with UTD control CART19 or CART22.

[0358] Figure 70Shown are the expressions of PD-L1, PD1, LAG3, and TIM3 (from left to right in each group of four bars) in lymph node and bone marrow samples from five CR patients, one unclassified patient, and six PD patients.

[0359] Figure 71 is a graph showing activation (in RLU) of several CD22 CAR constructs in the presence and absence of m971 competitor.

[0360] Figure 72 is a graph showing activation (in RLU) of additional CD22 CAR constructs.

[0361] Figure 73 Three bar graphs indicating CD22 CAR activity in the IFN-γ assay are shown.

[0362] Figure 74 Binding activity of CD22-64 and CD22-65 CARs is shown.

[0363] Figure 75 is a diagram mapping the epitopes bound by various CD22 scFvs.

[0364] Details

[0365] definition

[0366] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0367] The terms "a" and "an" refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element.

[0368] When referring to a measurable value such as an amount, a temporal duration, and the like, the term "about" is meant to encompass variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value, such that such variations are appropriate for performing the disclosed methods.

[0369] As used herein, the term "apheresis" refers to an art-recognized in vitro method by which blood from a donor or patient is removed from the donor or patient and passed through a device that separates selected specific components and returns the remainder to the donor's or patient's circulatory system, for example, by retransfusion. Thus, an "apheresis sample" refers to a sample obtained using apheresis.

[0370] The term "bioequivalent" refers to the amount of an active agent other than a reference compound (e.g., RAD001) that produces an effect equivalent to that produced by a reference dose or reference amount of a reference compound (e.g., RAD001). In one embodiment, the effect is the level of mTOR inhibition, for example, measured by P70 S6 kinase inhibition, for example, as assessed in an in vivo or in vitro assay, for example, by an assay as described herein, such as Boulay assay measurement, or by western blot measurement of phosphorylated S6 levels. In one embodiment, the effect is a change in the ratio of PD-1 positive / PD-1 negative T cells measured by cell sorting. In one embodiment, the bioequivalent amount or dosage of an mTOR inhibitor is an amount or dosage that achieves the same level of P70 S6 kinase inhibition as a reference dose or reference amount of a reference compound. In one embodiment, the bioequivalent amount or dosage of an mTOR inhibitor is an amount or dosage that achieves the same level of change in the ratio of PD-1 positive / PD-1 negative T cells as a reference dose or reference amount of a reference compound.

[0371] The term "inhibit" or "inhibitor" includes a decrease in certain parameters of a given molecule, such as CD20, CD10, CD19, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, such as activity. For example, inhibition of activity, such as inhibition of CD20, CD10, CD19, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a activity by at least 5%, 10%, 20%, 30%, 40% or more is included in this term. Thus, inhibition does not have to be 100%. The activity of the inhibitor can be determined as described herein or by assays known in the art. A "B cell inhibitor" is a molecule, such as a small molecule, an antibody, a CAR, or a cell comprising a CAR, which results in a decrease in a certain parameter, such as activity, such as growth or proliferation of B cells, or which results in a decrease in a certain parameter, such as the activity of a molecule associated with a B cell. Non-limiting examples of molecules associated with B cells include proteins expressed on the surface of B cells, such as CD20, CD10, CD19, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0372] The term "chimeric antigen receptor" or "CAR" refers to a group of polypeptides, generally two in the simplest embodiment, which, when in immune effector cells, provide cell specificity to target cells (usually cancer cells) and generate intracellular signals. In some embodiments, CAR comprises at least one extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which comprises a functional signaling domain derived from a stimulatory molecule and / or a co-stimulatory molecule as defined below. In some embodiments, the group of polypeptides is in the same polypeptide chain, for example, comprising a chimeric fusion protein. In some embodiments, the group of polypeptides is discontinuous with each other, for example, in different polypeptide chains. In some embodiments, the group of polypeptides includes a dimerization switch, which can couple polypeptides to each other in the presence of a dimerization molecule, for example, the antigen binding domain can be coupled to the intracellular signaling domain. On the one hand, the stimulatory molecule of CAR is a ζ chain bound to a T cell receptor complex (e.g., CD3ζ). In one aspect, the cytoplasmic transduction domain comprises a primary signaling domain (e.g., the primary signaling domain of CD3-ζ). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule defined below. On the one hand, costimulatory molecules are selected from costimulatory molecules as described herein, such as 4-1BB (i.e., CD137), CD27 and / or CD28. On the one hand, CAR comprises 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. On the one hand, CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. On the one hand, CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. On the one hand, CAR includes a chimeric fusion protein, which includes an extracellular antigen binding domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain includes at least two functional signal transduction domains derived from one or more costimulatory molecules and a functional signal transduction domain derived from stimulation. In one aspect, CAR includes an optional leader sequence on the amino terminus (N-ter) of the CAR fusion protein. In one aspect, CAR is also included in the leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cut from the antigen binding domain (e.g., scFv) during cell processing and locates CAR to the cell membrane.

[0373] As used herein, the phrase "disease associated with CD20 expression" includes, but is not limited to, diseases associated with CD20 (e.g., wild-type or mutant CD20) expression or conditions associated with CD20 (e.g., wild-type or mutant CD20) expressed or expressed at any time, including, for example, proliferative diseases such as cancer or malignancy or precancerous conditions such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or non-cancer-related indications associated with cells expressing CD20 (e.g., wild-type or mutant CD20). For the avoidance of doubt, diseases associated with CD20 expression may include conditions associated with cells that do not currently express CD20 but once expressed CD20, for example, because CD20 expression has been downregulated, for example, due to treatment with a molecule targeting CD20, e.g., CD20 CAR. In one aspect, the cancer associated with CD20 expression is a blood cancer. In one aspect, blood cancers include, but are not limited to, AML, myelodysplastic syndrome, ALL, hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, blastic plasmacytoid dendritic cell neoplasm, and the like. Other diseases associated with CD20 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with CD20 expression. Non-cancer related indications associated with CD20 expression may also be included. In some embodiments, CD20-expressing cells express or express CD20 mRNA at any time. In one embodiment, cells expressing CD20 produce CD20 protein (e.g., wild-type or mutant), which may be present at normal or reduced levels. In one embodiment, CD20-expressing cells produce detectable levels of CD20 protein at one point and subsequently produce substantially no detectable CD20 protein.

[0374] As used herein, the phrase "disease associated with CD22 expression" includes, but is not limited to, diseases associated with CD22 (e.g., wild-type or mutant CD22) expression or conditions associated with CD22 (e.g., wild-type or mutant CD22) expressed or expressed at any time, including, for example, proliferative diseases such as cancer or malignancies or precancerous conditions such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or non-cancer-related indications associated with cells expressing CD22 (e.g., wild-type or mutant CD22). For the avoidance of doubt, diseases associated with CD22 expression may include conditions associated with cells that do not currently express CD22 but once expressed CD22, for example, because CD22 expression has been downregulated, for example, due to treatment with a molecule targeting CD22, e.g., CD22 CAR. In one aspect, the cancer associated with CD22 expression is a blood cancer. In one aspect, blood cancers include, but are not limited to, AML, myelodysplastic syndrome, ALL, hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, blastic plasmacytoid dendritic cell tumors, and the like. Other diseases associated with CD22 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with CD22 expression. Non-cancer related indications associated with CD22 expression may also be included. In some embodiments, CD22-expressing cells express or express CD22 mRNA at any time. In one embodiment, cells expressing CD22 produce CD22 protein (e.g., wild-type or mutant), which may be present at normal or reduced levels. In one embodiment, CD22-expressing cells produce detectable levels of CD22 protein at one point and subsequently produce substantially no detectable CD22 protein.

[0375] As used herein, unless otherwise indicated, the terms "prevent," "preventing," and "suppressing" refer to an action that occurs before a subject begins to suffer from a condition or before a condition recurs. Preventing does not require complete prevention of a condition; the term includes partial prevention or alleviation of a condition or symptom of a condition, or reducing the risk of developing a condition.

[0376] As used herein, "combination" administration refers to delivering two (or more) different treatments to a subject while the subject is suffering from a disease, for example, delivering two or more treatments after the subject is diagnosed with the disease and before the disease has been cured or eliminated, or before treatment has stopped for other reasons. In some embodiments, when the delivery of the second treatment begins, the delivery of one treatment is still occurring, so that there is overlap in administration. This is sometimes referred to as "simultaneous" or "simultaneous delivery" in this article. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective due to the combined administration. For example, the second treatment is more effective, for example, the same effect can be seen with less of the second treatment than if the second treatment were administered without the first treatment, or compared to a similar situation seen with the first treatment, or the second treatment can alleviate symptoms to a greater extent. In some embodiments, delivery causes a greater alleviation of symptoms or other parameters related to the disease than the alleviation of symptoms observed when a treatment is delivered in the absence of another treatment. The effects of the two treatments can be partially additive, completely additive, or greater than additive. Delivery can be such that the effect of the first treatment delivered can still be detected when the second treatment is delivered. In one embodiment, the CAR expressing cells are administered at a dosage and / or dosing regimen as described herein, and a B cell inhibitor or active agent that enhances the activity of the CD19 CAR expressing cells is administered at a dosage and / or dosing regimen as described herein.

[0377] As used herein, the term "derived from" refers to a relationship between a first and a second molecule. It generally refers to the structural similarity between the first and second molecules and does not imply or include any limitation on the method or source of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure to have the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include any limitation on a particular method for generating the intracellular signaling domain. For example, it does not mean that to provide an intracellular signaling domain, one must start with the CD3ζ sequence and delete unwanted sequences, or impose mutations, in order to arrive at the intracellular signaling domain.

[0378] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate the activity of the cell through defined signaling pathways by generating second messengers or by acting as an effector in response to such messengers.

[0379] As used herein, the term "CD19" refers to the cluster of differentiation 19 protein, which is an antigenic determinant detectable on leukemic precursor 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" includes proteins comprising mutations, such as point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD19. CD19 is expressed in most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other cells expressing CD19 are provided below in the definition of "diseases associated with CD19 expression." It is also an early marker for B cell progenitors. See, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). On the one hand, the antigen-binding portion of CART recognizes and binds to an antigen within the extracellular domain of the CD19 protein. On the one hand, the CD19 protein is expressed on cancer cells.

[0380] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0381] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with the epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide bond-connected Fvs (sdFv), the Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAb (VL or VH), camelid VHH domains, multispecific antibodies formed by antibody fragments (e.g., a bivalent fragment comprising two Fab fragments connected by a disulfide bond in the hinge region) and the CDR or other epitope binding fragments of the separation of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maximal antibodies, miniantibodies, nanobodies, intrabodies, double antibodies, three antibodies, four antibodies, v-NARs, and double-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can also be grafted to polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0382] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguous via, for example, a synthetic linker, such as a short, flexible polypeptide linker, and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, scFv can have the VL and VH variable regions in either order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0383] As used herein, the term "complementarity determining region" or "CDR" refers to an amino acid sequence within an antibody variable region that confers antigen specificity and binding affinity. For example, generally, 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 any of a number of well-known schemes, including 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 a combination thereof. According to the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues 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 CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In the combined Kabat and Chothia numbering schemes, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH (e.g., a mammalian VH, e.g., a human VH); and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL (e.g., a mammalian VL, e.g., a human VL).

[0384] As used herein, the term "binding domain" or "antibody molecule" refers to a protein, such as an immunoglobulin chain or a fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" includes antibodies and antibody fragments. In one embodiment, the antibody molecule is a multispecific antibody molecule, for example, it comprises a plurality of immunoglobulin variable domain sequences, wherein the plurality of first immunoglobulin variable domain sequences have binding specificity to a first epitope and the plurality of second immunoglobulin variable domain sequences have binding specificity to a second epitope. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. The bispecific antibody molecule is characterized in that a first immunoglobulin variable domain sequence has binding specificity to a first epitope and a second immunoglobulin variable domain sequence has binding specificity to a second epitope.

[0385] The portion of the CAR of the present invention comprising an antibody or its antibody fragment can exist in various forms, wherein the antigen binding domain is expressed as a part of a continuous polypeptide chain, including, for example, 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 aspect, the antigen binding domain of the CAR composition of the present invention comprises an antibody fragment. On the other hand, CAR comprises an antibody fragment comprising scFv.

[0386] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in an antibody molecule in its naturally occurring configuration and which generally determines the class to which the antibody belongs.

[0387] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in an antibody molecule in its naturally occurring configuration. Kappa (κ) and lambda (λ) light chains refer to the two major isotypes of antibody light chains.

[0388] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a phage or yeast expression system. The term should also be interpreted as referring to an antibody that has been produced by synthesizing a DNA molecule encoding the antibody (and wherein the DNA molecule expresses the antibody protein) or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0389] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. The immune response may involve the production of antibodies or the activation of cells with specific immunocompetence, or both. Those skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Additionally, antigens may be derived from recombinant or genomic DNA. When using this term herein, those skilled in the art will appreciate that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, thus encoding an "antigen." Additionally, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit a desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It will be apparent that an antigen may be produced synthetically, or may be derived from a biological sample, or may be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or fluids having other biological components.

[0390] The terms "competition" or "cross-competition" are used interchangeably herein to refer to the ability of an antibody molecule to interfere with the binding of an antibody molecule (e.g., an anti-CD20 or CD22 antibody molecule provided herein) to a target (e.g., human CD20 or CD22). The interference with binding can be direct or indirect (e.g., by allosteric regulation of the antibody molecule or target). The degree to which an antibody molecule is able to interfere with the binding of another antibody molecule to a target, and therefore whether the degree of competition can be determined using, for example, a competitive binding assay as described herein, can be used. In some embodiments, the competitive binding assay is a quantitative competition assay. In some embodiments, a first antibody molecule is said to compete for binding to a target with a second antibody molecule when binding of the first antibody molecule to the target is reduced by 10% or more, e.g., 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, 99% or more in a competition binding assay (e.g., a competition assay described herein).

[0391] As used herein, the term "epitope" refers to a portion of an antigen (e.g., human CD20 or CD22) that specifically interacts with an antibody molecule. These portions, referred to herein as epitope determinants, typically comprise elements 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, such as by crystallography or by hydrogen-deuterium exchange. At least one or some portions of an antibody molecule that specifically interact with an epitope determinant are typically located in a CDR. Typically, an epitope has specific three-dimensional structural characteristics. Typically, an epitope has specific charge characteristics. Some epitopes are linear epitopes, while other epitopes are conformational epitopes.

[0392] The term "anti-cancer effect" refers to a biological effect that can be manifested by a variety of means, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with cancerous conditions. An "anti-cancer effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies described herein to prevent the development of cancer in the first place. The term "anti-tumor effect" refers to a biological effect that can be manifested by a variety of means, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0393] The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual.

[0394] The term "allogeneic" refers to any material derived from an animal of the same species as the individual into which the material is to be introduced. Two or more individuals are considered allogeneic to one another when the genes at one or more loci are not identical. In certain aspects, allogeneic material from individuals of the same species can be sufficiently genetically distinct to interact antigenically.

[0395] The term "xenogeneic" means that the transplant originates from a different species of animal.

[0396] The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, for example, and the two terms include solid and liquid, for example, diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include pre-malignant, as well as malignant cancers and tumors.

[0397] The terms "cancer-associated antigen" or "tumor antigen" or "proliferative disorder antigen" or "antigen associated with a proliferative disorder" interchangeably refer to a molecule (typically a protein, carbohydrate, or lipid) that is preferentially expressed on the surface of cancer cells, either in whole or in fragment form (e.g., MHC / peptide), compared to normal cells, and is used to preferentially target pharmacological agents to cancer cells. In some embodiments, a tumor antigen is a marker expressed by normal and cancer cells, such as a lineage marker such as CD19 on B cells. In certain aspects, the tumor antigens of the present invention are derived from cancers, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like. In some embodiments, a tumor antigen is an antigen common to specific proliferative disorders. In some embodiments, the antigen associated with cancer is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, for example, 1 times overexpression, 2 times overexpression, 3 times or more overexpression compared to normal cells. In some embodiments, the antigen associated with cancer is a cell surface molecule that is inappropriately synthesized in cancer cells, for example, a molecule containing deletions, additions or mutations compared to molecules expressed on normal cells. In some embodiments, the antigen associated with cancer should be specifically expressed on the cell surface of cancer cells in whole or in fragment form (for example, MHC / peptides), rather than synthesized or expressed on the surface of normal cells. In some embodiments, the CAR of the present invention includes a CAR comprising an antigen binding domain (for example, an antibody or antibody fragment) that binds to an MHC presenting peptide. Typically, peptides derived from endogenous proteins fill the pockets of class I molecules of the major histocompatibility complex (MHC) and are recognized by T cell receptors (TCRs) on CD8+T lymphocytes. Class I MHC complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy.TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 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 20135(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified from screening libraries such as human scFv phage display libraries.

[0398] The phrase "disease associated with CD19 expression" includes, but is not limited to, diseases associated with CD19 (e.g., wild-type or mutant CD19) expression or disorders associated with cells expressing or expressing CD19 (e.g., wild-type or mutant CD19) at any time, including, for example, proliferative diseases such as cancer or malignancies or precancerous conditions such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or non-cancer-related indications associated with cells expressing CD19. For the avoidance of doubt, diseases associated with CD19 expression may include disorders associated with cells that currently do not express CD19, for example because CD19 expression has been downregulated, for example due to treatment with molecules targeting CD19, for example, CD19 CAR, but previously expressed CD19. On the one hand, the cancer associated with the expression of CD19 is a blood cancer. On the one hand, a blood cancer is a leukemia or lymphoma. In one aspect, cancers associated with expression of CD19 include cancers and malignancies, including but not limited to, for example, one or more acute leukemias, including but not limited to, for example, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias, including but not limited to chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Additional cancers or hematologic disorders associated with CD19 expression include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia," a diverse group of hematologic conditions associated with ineffective production (or dysplasia) of myeloid blood cells, etc. Other diseases associated with CD19 expression include, but are not limited to, atypical and / or nonclassical cancers, malignancies, precancerous conditions, or proliferative diseases associated with CD19 expression. Non-cancer related indications associated with CD19 expression include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory diseases (allergy and asthma), and transplantation. In some embodiments, CD19 expressing cells express or express CD19 mRNA at any time. In one embodiment, cells expressing CD19 produce CD19 protein (e.g., wild type or mutant), and the CD19 protein can be present at normal levels or reduced levels. In one embodiment, cells expressing CD19 produce detectable levels of CD19 protein at one point and then substantially do not produce detectable CD19 protein.

[0399] The term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody or antibody fragment containing the 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 art (such as site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-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 within the CAR of the present invention can be replaced by amino acid residues from the same side chain family, and the changed CAR can be tested using functional assays as described herein.

[0400] The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex or CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex or signal transduction through the appropriate NK receptor or signaling domain of the CAR. Stimulation can mediate altered expression of certain molecules.

[0401] The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., a T cell, NK cell, or B cell) that provides a cytoplasmic signaling sequence that regulates immune cell activation in a stimulatory manner for at least some aspects of the immune cell signaling pathway. In one aspect, the signal is initiated by, for example, the binding of a TCR / CD3 complex to a peptide-loaded MHC molecule, and results in the primary signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. The primary cytoplasmic signaling sequence that acts in a stimulatory manner (also referred to as a "primary signaling domain") may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of cytoplasmic signaling sequences containing ITAMs that are particularly useful in the present invention include, but are not limited to, those derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In the specific CARs of the present invention, the intracellular signaling domain in any one or more CARs of the present invention includes an intracellular signaling sequence, such as the primary signaling sequence of CD3-ζ. In the specific CARs of the present invention, the primary signaling sequence of CD3-ζ is a sequence as provided in SEQ ID NO: 17 or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc. In the specific CARs of the present invention, the primary signaling sequence of CD3-ζ is a sequence as provided in SEQ ID NO: 43 or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.

[0402] The term "antigen presenting cell" or "APC" refers to a cell of the immune system, such as a helper cell (e.g., B-cell, dendritic cell, etc.), that presents foreign antigens complexed with major histocompatibility complexes (MHCs) on its surface. T-cells can recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

[0403] The term "immune effector cell" as used herein refers to a cell that participates in an immune response, e.g., participates in promoting an immune effector response. Examples of immune effector cells include T cells, e.g., α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NK-T) cells, mast cells, and bone marrow-derived phagocytes.

[0404] As used herein, the term "immune effector function or immune effector response" refers to a function or response of an immune effector cell that, for example, enhances or promotes an immune attack on a target cell. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes killing of a target cell or inhibits its growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0405] The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines.

[0406] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can produce signals that promote the immune effector function of CAR-containing cells, such as CART cells. For example, examples of immune effector functions in CART cells include cytolytic activity and auxiliary activity, including the secretion of cytokines. In an embodiment, the intracellular signaling domain is part of a protein that transduces effector function signals and guides cells to perform specialized functions. Although the entire intracellular signaling domain can be used, in many cases, it is not necessary to use the entire chain. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used to replace the complete chain, as long as it transduces the effector function signal. The term intracellular signaling domain is therefore intended to include any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal.

[0407] In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may include a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may include a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may include a cytoplasmic sequence from a common receptor or a costimulatory molecule.

[0408] The primary intracellular signaling domain can include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from: CD3ζ, FcRγ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, CD32, DAP10, and DAP12.

[0409] The term "ζ" or alternatively "ζ chain", "CD3-ζ" or "TCRζ" is defined as the protein as provided in GenBank Acc. No. BAG36664.1 or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.), and a "ζ stimulatory domain" or alternatively "CD3-ζ stimulatory domain" or "TCR-ζ stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the ζ chain or a functional derivative thereof that are sufficient to functionally transmit the initiation signal required for T cell activation. In one aspect, the cytoplasmic domain of ζ includes residues 52 to 164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) that are functional homologs thereof. In one aspect, the "ζ stimulatory domain" or "CD3-ζ stimulatory domain" is the sequence provided in SEQ ID NO: 17. In one aspect, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:43.

[0410] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that promote an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating 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 (CD27 8),GITR,BAFFR,LIGHT,HVEM(LIGHTR),KIRDS2,SLAMF7,NKp80(KLRF1),NKp44,NKp30,NKp46,CD19,CD4,C D8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, 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 ,NKG2D,NKG2C,TNFR2,TRANCE / RANKL,DNAM1(CD226),SLAMF4(CD244,2B4),CD84,CD96(Tactile),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 ligands that specifically bind to CD83.

[0411] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain can include the entire intracellular portion of a molecule or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0412] The term "4-1BB" refers to a member of the TNFR superfamily having an amino acid sequence as provided in GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.; and the "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. In one aspect, the "4-1BB costimulatory domain" is a sequence as provided in SEQ ID NO: 16, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.

[0413] The term "coding" refers to the inherent properties of the specific sequence of the nucleotides in polynucleotides such as genes, cDNAs or mRNAs as templates for synthesizing other polymers and macromolecules in biological processes, and this polymer and macromolecule have definite nucleotide sequence (for example, rRNA, tRNA and mRNA) or definite amino acid sequence and the biological properties obtained therefrom. Therefore, if the transcription and translation of the mRNA corresponding to the gene produce protein in cells or other biological systems, then gene, cDNA or RNA encodes protein. Its nucleotide sequence is identical to the mRNA sequence and is usually provided in the coding strand in the sequence table and the non-coding strand as the template for transcribed gene or cDNA, both of which can be referred to as the protein or other products of the gene or cDNA that encodes.

[0414] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some forms.

[0415] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance or composition, as described herein, effective to achieve a particular biological result.

[0416] The term "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0417] The term "exogenous" refers to any substance introduced or generated from outside an organism, cell, tissue or system.

[0418] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0419] The term "transfer vector" refers to a composition of matter that includes an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. A large number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Therefore, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to further include non-plasmids and non-viral compounds that promote the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0420] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which the recombinant polynucleotide is incorporated.

[0421] The term "lentivirus" refers to a species of the retrovirus family. Lentiviruses are unique among retroviruses because they can infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0422] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, particularly including self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8): 1453–1464 (2009). Other examples of lentiviral vectors that can be used in the clinic include, but are not limited to, for example, lentiviral vectors from Oxford BioMedica. Gene delivery technology from Lentigen Vector Systems, etc. Non-clinical varieties of lentiviral vectors are also available and will be known to those skilled in the art.

[0423] The term "homologous" or "identity" refers to the subunit sequence identity between two polymeric molecules, for example, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit site in the two molecules is occupied by the same monomeric subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the sites in the two sequences (for example, five sites in a polymer that is ten subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the sites (for example, 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0424] The form of "humanized" non-human (e.g., murine) antibodies is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) containing a minimal sequence derived from a non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibodies or antibody fragments) in which the residues from the complementary determining regions (CDRs) of the recipient are replaced by residues from the CDRs of non-human species (donor antibodies) such as mice, rats, or rabbits with the desired specificity, affinity, and ability. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies / antibody fragments can include residues that are neither found in the recipient antibody nor found in the introduced CDR or framework sequences. These modifications can further improve and optimize antibody or antibody fragment performance. Typically, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also include at least a portion of an immunoglobulin constant region (Fc), typically those 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.

[0425] "Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, wherein the overall molecule is of human origin or consists of the same amino acid sequence as the human form of the antibody or immunoglobulin.

[0426] The term "isolated" means altered or removed from its native state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its native state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment such as, for example, a host cell.

[0427] In the context of the present invention, the following commonly occurring abbreviations for 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.

[0428] The term "operably linked" or "transcriptional control" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous to each other and, for example, must be in the same reading frame when joining two protein coding regions.

[0429] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, intratumoral or infusion techniques.

[0430] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. The term "nucleic acid" includes genes, cDNA or mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless explicitly defined, the term includes nucleic acids containing analogs or derivatives of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as clearly specified sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which three positions of one or more selected (or all) codons are substituted with mixed-base and / or deoxyinosine residues (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)).

[0431] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds consisting 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 comprise a sequence of a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids bound to each other by peptide bonds. As used herein, the term refers to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) as well as longer chains (which are also commonly referred to in the art as proteins, of which there are many types). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0432] The term "promoter" refers to a DNA sequence that is recognized by, or introduced into, the synthetic machinery of the cell and is required to initiate specific transcription of a polynucleotide sequence.

[0433] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product to which the promoter / regulatory sequence is operably linked. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be a sequence that expresses the gene product in a tissue-specific manner.

[0434] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0435] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

[0436] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene, causes the gene product to be produced in a cell substantially only when the cell is of the tissue type corresponding to the promoter.

[0437] As used in the context of scFv, the term "flexible polypeptide linker" or "linker" refers to a peptide linker composed of amino acids such as glycine and / or serine residues, used alone or in combination, that connects the variable heavy chain and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser) n，Wherein 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, the flexible polypeptide linker includes but is not limited to (Gly4Ser)4 (SEQ ID NO: 106) or (Gly4Ser)3 (SEQ ID NO: 107). In another embodiment, the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO: 108). Also included within the scope of the present invention are the linkers described in WO2012 / 138475, which is incorporated herein by reference.

[0438] As used herein, the 5' cap (also known as the RNA cap, RNA 7-methylguanosine cap, or RNA m 7 The G cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of a eukaryotic messenger RNA shortly after transcription begins. The 5' cap consists of a terminal group connected to the first transcribed nucleotide. Its presence is important for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription and is performed in an auxiliary transcription manner so that each affects the other. Shortly after transcription begins, the 5' end of the synthesized mRNA is bound by a cap synthesis complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis is carried out in a multi-step biochemical reaction manner. The capping portion can be modified to regulate the functionality of the mRNA, such as its translation stability or efficiency.

[0439] As used herein, "in vitro transcribed RNA" refers to RNA that has been synthesized in vitro, e.g., mRNA. Typically, in vitro transcribed RNA is produced by an in vitro transcription vector. The in vitro transcription vector comprises a template for producing in vitro transcribed RNA.

[0440] As used herein, "poly (A)" is a series of adenosines attached to an mRNA by polyadenylation. In some embodiments of transiently expressed constructs, the poly A is between 50 and 5000 (SEQ ID NO: 28), such as greater than 64, such as greater than 100, such as greater than 300 or 400. The poly (A) sequence can be modified chemically or enzymatically to modulate mRNA function, such as localization, stability, or translation efficiency.

[0441] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenosine moiety or its modified variants to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (usually several hundred) added to the pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts containing a specific sequence (polyadenylation signal). The poly(A) tail and the proteins that bind to it help protect the mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after DNA is transcribed into RNA, but can also occur later in the cytoplasm. After transcription has terminated, the mRNA chain is cleaved by the action of an endonuclease complex bound to RNA polymerase. The cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0442] As used herein, "transient" refers to the expression of a non-integrated transgene for a period of hours, days, or weeks, where the period of expression is less than the period of expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0443] The term "signal transduction pathway" refers to the biochemical relationships between various signal transduction molecules that play a role in transducing a signal from one part of a cell to another. The phrase "cell surface receptor" includes molecules and molecular complexes that are capable of receiving and transmitting signals across the cell membrane.

[0444] The term "subject" is meant to include living organisms (eg, mammals, humans) in which an immune response can be elicited.

[0445] The term "substantially purified" cells refers to cells that are substantially free of other cell types. Substantially purified cells refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some cases, a substantially purified cell population refers to a homogenous population of cells. In other cases, the term refers only to cells that have been separated from cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0446] As used herein, the term "treat" means to treat. A therapeutic effect is achieved by alleviating, suppressing, alleviating or eradicating the disease state.

[0447] As used herein, the term "prevention" refers to the prophylactic or protective treatment of a disease or disease state.

[0448] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "hyperproliferative disorder-associated antigen" refers to an antigen that is common to a particular hyperproliferative disease. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from cancer, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.

[0449] The term "transfected" or "transformed" or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include primary subject cells and their progeny.

[0450] A subject "responds" to treatment if a parameter of the cancer (e.g., a hematologic cancer, such as cancer cell growth, proliferation, and / or survival) in the subject is delayed or reduced by a detectable amount, such as about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (as measured by any appropriate measurement, such as mass, cell count, or volume). In one example, a subject responds to treatment if the subject experiences a life expectancy that is about 5%, 10%, 20%, 30%, 40%, 50% or more longer than the life expectancy that would have occurred had the treatment not been administered. In another example, a subject responds to treatment if the subject has increased disease-free survival, overall survival, or increased time to progression. Several methods can be used to determine whether a patient responds to treatment, including, for example, the NCCN Clinical Practice Guidelines in Oncology (NCCN ) provided by the criteria. For example, in the context of B-ALL, a complete response or complete responder may involve one or more of the following: <5% BM blasts, >1000 neutrophils / ANC ( / μL), >100,000 platelets ( / μL), no circulating blasts or extramedullary disease (no lymphadenopathy, splenomegaly, skin / gingival infiltration / testicular mass / CNS involvement), third-line hematopoiesis, and no relapse for 4 weeks. A partial responder may involve one or more of >50% reduction in BM blasts, >1000 neutrophils / ANC ( / μL), >100,000 platelets ( / μL). A non-responder may show disease progression, such as >25% in BM blasts.

[0451] As used herein, "refractory" refers to a disease, such as cancer, that is unresponsive to treatment. In 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 cancers are also known as drug-resistant cancers.

[0452] The term "relapse" as used herein refers to the recurrence of cancer after the initial response period (e.g., complete response or partial response). The initial response period may involve cancer cell levels below a certain threshold, for example, below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Recurrence may involve cancer cells rising to a level above a certain threshold, for example, above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, recurrence may involve, for example, the recurrence of blasts in blood, bone marrow (>5%) or any extramedullary site after a complete response. In this case, a complete response may involve <5% BM blasts. More generally, in one embodiment, a response (e.g., complete response or partial response) may involve 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.

[0453] In some embodiments, the therapy including CD19 inhibitors (such as CD19 CAR therapy) may relapse or be refractory to treatment. Relapse or resistance can be caused by CD19 loss (for example, antigen loss mutation) or other CD19 changes that reduce CD19 levels (for example, caused by clonal selection of CD19 negative clones). Cancers carrying such CD19 losses or changes are referred to herein as "CD19 negative cancers" or "CD19 negative recurrent cancers"). It should be understood that CD19 negative cancers do not require 100% CD19 loss, but are sufficient to reduce the effectiveness of CD19 therapy, causing cancer to relapse or become refractory. In some embodiments, CD19 negative cancers are from CD19 CA therapy.

[0454] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (eg, a stimulatory tumor antigen) present in a sample, but does not substantially recognize or bind other molecules in the sample.

[0455] As used herein, the term "pharmaceutically acceptable salt" refers to salts that are suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, etc., within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1–19.

[0456] The term "regulatable chimeric antigen receptor (RCAR)" as used herein refers to a group of polypeptides, typically two polypeptides in the simplest embodiment, which, when in RCARX cells, provide RCARX cells with specificity for target cells, typically cancer cells, and have regulatable intracellular signal generation or proliferation that can optimize the immune effector properties of RCARX cells. RCARX cells rely at least in part on the antigen binding domain to provide specificity to target cells comprising an antigen bound by the antigen binding domain. In one embodiment, RCAR includes a dimerization switch that can couple the intracellular signaling domain to the antigen binding domain in the presence of a dimerization molecule.

[0457] As used herein, the term "membrane anchor" or "membrane tethering domain" refers to a polypeptide or a moiety, such as a myristyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

[0458] As used herein, the term "switch domain", for example, when it relates to RCAR, refers to an entity associated with another switch domain in the presence of a dimerization molecule, generally an entity based on polypeptide. The association results in the functional coupling of a first entity connected (such as fused) to the first switch domain and a second entity connected (such as fused) to the second switch domain. The first and second switch domains are together referred to as dimerization switches. In embodiments, the first and second switch domains are identical to each other, for example, they are polypeptides with identical primary amino acid sequences, and are together referred to as homodimerization switches. In embodiments, the first and second switch domains are different from each other, for example, they are polypeptides with different primary amino acid sequences, and are together referred to as heterodimerization switches. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is an entity based on polypeptide, for example, based on FKBP or FRB, and the dimerization molecule is a small molecule, such as rapalog. In an embodiment, the switch domain is a polypeptide-based entity, such as a scFv that binds a myc peptide, and the dimerization molecule is a polypeptide, a fragment thereof, or a multimer of polypeptides, such as a myc ligand or a multimer of myc ligands that binds one or more myc scFvs. In an embodiment, the switch domain is a polypeptide-based entity, such as a myc receptor, and the dimerization molecule is an antibody or a fragment thereof, such as a myc antibody.

[0459] As used herein, the term "dimerization molecule", for example, when referring to RCAR, refers to a molecule that promotes the binding of the first switch domain to the second switch domain. In embodiments, the dimerization molecule is not naturally present in the subject, or is not present at a concentration that results in significant dimerization. In embodiments, the dimerization molecule is a small molecule, such as rapamycin or rapamycin series, for example, RAD001.

[0460] When used in combination with an mTOR inhibitor (e.g., an allosteric mTOR inhibitor, such as RAD001 or rapamycin or a catalytic mTOR inhibitor), the term "low immune enhancing dose" refers to a dose of an mTOR inhibitor that partially, but not completely, inhibits mTOR activity, for example, as measured by inhibiting P70 S6 kinase activity. Methods for assessing mTOR activity (e.g., by inhibiting P70 S6 kinase) are discussed herein. The dose is not sufficient to cause complete immunosuppression, but is sufficient to enhance the immune response. In one embodiment, a low immune enhancing dose of an mTOR inhibitor results in 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 ratio of PD-1 negative T cells to PD-1 positive T cells. In one embodiment, a low immune enhancing dose of an mTOR inhibitor results in an increase in the number of naive T cells. In one embodiment, a low immune enhancing dose of an mTOR inhibitor results in one or more of the following:

[0461] For example, increased expression of one or more of the following markers on memory T cells, such as memory T cell precursors: CD62L 高 ,CD127 高 ,CD27 + and BCL2,;

[0462] For example, reduced expression of KLRG1 on memory T cells, such as memory T cell precursors; and

[0463] Increased numbers of memory T cell precursors, such as cells with any one or a combination of the following characteristics: CD62L 高 Increased CD127 高 Increased CD27 + increased, KLRG1 decreased and BCL2 increased;

[0464] wherein any of the above-mentioned changes in presence occur, for example, at least transiently, for example, as compared to an untreated subject.

[0465] Ranges: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as from 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes ranges having 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the breadth of the range.

[0466] describe

[0467] CD19 inhibitors and binding domains

[0468] Provided herein are compositions of matter and methods of use for treating diseases such as cancer using CD19 chimeric antigen receptors (CARs). The methods particularly include administering in combination CD19 CARs as described herein with another active agent such as a B cell inhibitor. The methods also include, for example, administering CD19 CARs as described herein to treat lymphomas, such as Hodgkin's lymphoma.

[0469] On the one hand, the invention provides many chimeric antigen receptors (CARs), which include antibodies or antibody fragments engineered for specific binding to CD19 proteins. On the one hand, the invention provides cells (e.g., T cells) engineered to express CAR, wherein CAR T cells ("CART") show anti-cancer properties. In one aspect, cells are transformed with CAR and expressed on the cell surface. In some embodiments, cells (e.g., T cells) are transduced with viral vectors encoding 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 CAR. In another embodiment, cells (e.g., T cells) are transfected with nucleic acids encoding CAR, such as mRNA, cDNA, DNA. In some such embodiments, cells can transiently express CAR.

[0470] On the one hand, the anti-CD19 protein binding portion of CAR is a scFv antibody fragment. In one aspect, such antibody fragments are functional because they retain equivalent binding affinity, for example, they bind to the same antigen with an affinity comparable to that of the IgG antibody from which they are derived. In one aspect, such antibody fragments are functional because they provide a biological response, which may include but is not limited to the activation of an immune response, inhibition of signal transduction from its target antigen, inhibition of kinase activity, etc., as will be understood by those skilled in the art. On the one hand, the anti-CD19 antigen binding domain of CAR is a humanized scFv antibody fragment compared to the mouse sequence of the scFv from which it is derived. On the one hand, the parent mouse scFv sequence is the CAR19 construct provided in PCT Publication WO2012 / 079000 and is provided herein as SEQ ID NO: 59. In one embodiment, the anti-CD19 binding domain is a scFv described in WO2012 / 079000 and provided in SEQ ID NO: 59 or a sequence having at least 95%, for example 95-99% identity thereto. In one embodiment, the anti-CD19 binding domain is a part of the CAR construct provided in PCT Publication WO2012 / 079000 and is provided herein as SEQ ID NO: 58, or a sequence having at least 95%, for example 95%-99% identity thereto. In one embodiment, the anti-CD19 binding domain comprises at least one (e.g., 2, 3, 4, 5 or 6) CDR selected from Table 4 and / or Table 5.

[0471] In some aspects, the antibody of the present invention is incorporated into a chimeric antigen receptor (CAR). On the one hand, CAR is included in PCT Publication WO2012 / 079000 as SEQ ID NO: 12 is provided and herein as SEQ ID NO: 58 provides a polypeptide sequence, wherein the scFv domain is selected from one or more SEQ ID NOS: 1-12 sequence replacements. On the one hand, SEQ ID NO: 1-12 scFv domains are SEQ ID NO: 59 scFv domains humanized variants, which are scFv fragments of mouse origin specifically binding to human CD19. This mouse scFv humanization may be desirable for clinical settings, where mouse-specific residues can be induced in patients receiving CART19 treatment (e.g., treatment with T cells transduced with CAR19 constructs) to respond to human anti-mouse antigens (HAMA).

[0472] In one aspect, the anti-CD19 binding domains of the CAR of the present invention, such as humanized scFv, are partially encoded by a transgenic gene, the sequence of which has been codon-optimized for expression in mammalian cells. In one aspect, the entire CAR construct of the present invention is encoded by a transgenic gene to which all sequences have been codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons encoding the same amino acids) in coding DNA is deviated in different species. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Various codon optimization methods are known in the art, including, for example, methods disclosed in at least US Patent Nos. 5,786,464 and 6,114,148.

[0473] In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 1. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 2. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 3. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 4. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 5. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 6. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 7. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 8. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 9. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 10. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 11. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 12.

[0474] On the one hand, the CAR of the present invention combines the antigen binding domain of a specific antibody with an intracellular signaling molecule. For example, in some aspects, intracellular signaling molecules include but are not limited to CD3-ζ chain, 4-1BB and CD28 signaling modules and combinations thereof. On the one hand, the CD19 CAR comprises a CAR selected from the sequence provided in one or more of SEQ ID NOs: 31-42. On the one hand, the CD19 CAR comprises the sequence provided in SEQ ID NO: 31. On the one hand, the CD19 CAR comprises the sequence provided in SEQ ID NO: 32. On the one hand, the CD19 CAR comprises the sequence provided in SEQ ID NO: 33. On the one hand, the CD19 CAR comprises the sequence provided in SEQ ID NO: 34. On the one hand, the CD19 CAR comprises the sequence provided in SEQ ID NO: 35. On the one hand, the CD19 CAR comprises the sequence provided in SEQ ID NO: 36. On the one hand, the CD19 CAR comprises the sequence provided in SEQ ID NO: 37. On the one hand, the CD19 CAR comprises the sequence provided in SEQ ID NO: 38. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO: 39. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO: 40. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO: 41. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO: 42.

[0475] Thus, in one aspect, the antigen binding domain comprises a humanized antibody or antibody fragment. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a murine or humanized anti-CD19 binding domain described herein, and / or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a murine or humanized anti-CD19 binding domain described herein, e.g., a humanized anti-CD19 binding domain comprising one or more, e.g., all three LC CDRs and one or more, e.g., all three HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a murine or humanized anti-CD19 binding domain described herein, e.g., the humanized anti-CD19 binding domain has two variable heavy chain regions, each variable heavy chain region comprising HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the humanized anti-CD19 binding domain comprises a humanized light chain variable region described herein (e.g., in Table 2) and / or a humanized heavy chain variable region described herein (e.g., in Table 2). In one embodiment, the humanized anti-CD19 binding domain comprises a humanized heavy chain variable region described herein (e.g., in Table 2), e.g., at least two humanized heavy chain variable regions described herein (e.g., in Table 2). In one embodiment, the anti-CD19 binding domain is an scFv comprising a light chain and a heavy chain having an amino acid sequence of Table 2. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided in Table 2, or a sequence with 95-99% identity with an amino acid sequence of Table 2; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided in Table 2, or a sequence with 95-99% identity with an amino acid sequence of Table 2.In one embodiment, the humanized anti-CD 19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or a sequence with 95-99% identity thereof....

Claims

1. Use of an effective amount of one or more cells expressing CAR molecules that bind to CD19 in the preparation of a medicament for treating a subject with a disease associated with CD19 expression in combination with one or more B cell inhibitors, wherein the B cell inhibitor comprises an inhibitor of one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

2. Use of a B cell inhibitor, such as an inhibitor of one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, in the preparation of a medicament for treating a subject, wherein the subject is or is identified as being a non-responder, partial responder, or relapser to a CD19 inhibitor, such as a CD19 CAR therapy.

3. A composition, wherein: (1) The composition comprises separately or mixed: (i) expressing one or more cells that bind to a CAR molecule that binds to CD19, such as a CAR molecule that binds to CD19 described herein, such as a CD19 CAR, and (ii) one or more B cell inhibitors selected from inhibitors of one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, or ROR1; or (2) The composition comprises: (i) a first nucleic acid encoding a CAR molecule that binds to CD19, e.g., a CAR molecule that binds to CD19 described herein, e.g., a CD19 CAR, and (ii) a second nucleic acid encoding a CAR molecule that binds to a B cell antigen selected from one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a, wherein the first nucleic acid and the second nucleic acid are in the same or separate nucleic acid molecules; or (3) The composition comprises one or more immune effector cells and, (i) a first nucleic acid or a first polypeptide encoding a first polypeptide, wherein the first polypeptide comprises a CAR molecule that binds to CD19, e.g., a CAR molecule that binds to CD19 described herein, e.g., a CD19 CAR, and (ii) a second nucleic acid or a second polypeptide encoding a second polypeptide, wherein the second polypeptide comprises 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.

4. A use of a CAR molecule that binds to CD19, the use being: (a) Use of a CAR molecule that binds to CD19, such as a CD19 CAR in combination with a PD1 inhibitor for the preparation of a medicament for treating a subject having DLBCL, e.g., primary DLBCL, wherein optionally the subject has or is identified as having at least 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of DLBCL cells that are CD3+ / PD1+; (b) use of a CAR molecule that binds to CD19, e.g., a CD19 CAR, for the preparation of a medicament for treating a subject having DLBCL, e.g., primary DLBCL, wherein optionally the subject has or is identified as having at least 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of DLBCL cells that are CD3+ / PD1+; or (c) Use of a CAR molecule that binds to CD19, such as a CD19 CAR, in combination with a PD1 inhibitor for the preparation of a medicament for treating a subject with DLBCL, wherein optionally the subject has or is identified as having less than 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of cancer, such as cells in the cancer microenvironment that are double positive for CD19 and PD-L1.

5. An isolated CAR molecule or an isolated nucleic acid molecule encoding said CAR, wherein: (1) the CAR comprises an antibody or antibody fragment, the antibody or antibody fragment comprising a CD20 binding domain, a transmembrane domain and an intracellular signaling domain, and wherein the CD20 binding domain comprises one or more light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2) and light chain complementary determining region 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 complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2) and heavy chain complementary determining region 3 (HC CDR3) of any CD20 heavy chain binding domain amino acid sequence listed in Tables 12A, 12B, 14A, or 14B; or (2) The CAR comprises an antibody or antibody fragment, which includes a CD22 binding domain, a transmembrane domain and an intracellular signaling domain, and wherein the CD22 binding domain comprises one or more light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2) and light chain complementary determining region 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 complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2) and heavy chain complementary determining region 3 (HC CDR3) of any CD22 heavy chain binding domain amino acid sequence listed in Tables 7A, 7B, 7C, 9A, or 9B.

6. A cell comprising the CAR-encoding nucleic acid or CAR molecule of claim 5.

7. Use of the cell containing the CAR molecule according to claim 6 in the preparation of a medicament for treating diseases related to CD20 or CD22 expression.

8. Use of an effective amount of a population of cells comprising a CAR molecule (CAR-expressing cells) in the preparation of a medicament for treating a subject having cancer, wherein the CAR molecule comprises a CD19 binding domain and a CD22 binding domain, wherein: (i) the CD19 binding domain comprises an scFv comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2 and HC CDR3 of FMC63; and (ii) the CD22 binding domain comprises an scFv, and the scFv comprises LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2 and HC CDR3 of m971; Optionally among them: (i) the subject is a young or pediatric subject; (ii) the subject has minimal residual disease; (iii) the subject has received a lymphodepletion regimen of cyclophosphamide and fludarabine prior to administration of the CAR-expressing cells; (iv) the subject has received stem cell transplantation before the administration of the CAR-expressing cells; (v) the subject has relapsed cancer (e.g., relapsed ALL); or (vi) The CAR-expressing cells are formulated to be administered for about 10 4 cells / kg to approximately 10 9 The dose was cells / kg.

9. Use of one or more B cell inhibitors in the preparation of a medicament for preventing CD19-negative relapse in a mammal, wherein the B cell inhibitor comprises an inhibitor of one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

10. An in vitro or ex vivo method or assay for identifying a subject having cancer who has an increased or decreased likelihood of responding to a treatment comprising a chimeric antigen receptor (CAR) therapy, the method comprising: (1) obtaining a sample from a subject; (2) Determine the value of one or more of the following: (i) the level of one or more markers listed in Table 29 in the sample; (ii) a characteristic of CD19, such as a mutation, such as a mutation resulting in a frameshift or premature stop codon or both, or (iii)T REG The level or activity of cells; and (3) optionally, comparing the determined value, e.g., the level, activity or property of (i), (ii) or (iii) or a combination thereof, to a reference value, wherein a difference, e.g., a statistically significant difference, between the determined value and the reference value predicts the subject's responsiveness to CAR therapy; and (4) Based on the determined value, the subject is identified as a complete responder, partial responder or non-responder to CAR therapy, or a relapser or non-relapser.

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