Systems and methods for monitoring the clonality and persistence of adoptive cell therapy.

Abstract

Methods and systems for identifying clinically effective populations of tumor-infiltrating lymphocytes are disclosed. Methods for identifying persistent unique clones derived from adoptive cell therapy products are also disclosed.

Description

[Technical Field]

[0001] Cross-reference of related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 790,898 filed on 10 January 2019, U.S. Provisional Patent Application No. 62 / 826,209 filed on 8 April 2019, and U.S. Provisional Patent Application No. 62 / 896,354 filed on 5 September 2019, all of which are incorporated herein by reference in their entirety.

[0002] Field of the present invention

[0002] The inventions described herein generally relate to identifying clinically effective populations of tumor-infiltrating lymphocytes, and more specifically, but not limited to, identifying clinically effective populations of T cells. [Background technology]

[0003] Background of the present invention

[0003] While general methods for profiling T cell receptors in cancer patients are known, e.g., Kirsch et al., Molecular Oncology, 9:2063-2070 (2015), these methods have proven to be of little use in evaluating the potential clinical response to cancer therapy. In particular, such profiling studies yield a multitude of data that do not draw clear conclusions regarding the effect of specific therapies on T cell diversity and the relationship between measured diversity and clinical response and / or clinical outcome, e.g., Snyder et al., PLOS Medicine, 14:e1002309 (2017). This difficulty is particularly serious with adoptive cell therapies that themselves alter the patient's immune repertoire, e.g., Milone and Bhoj, Molecular Therapy: Methods & Clinical Development 8:210-221 (2018).

[0004]

[0004] In particular, in adoptive cell therapy, the identification of clinically relevant subpopulations of cells within a heterogeneous polyclonal therapeutic population of cells is an important issue. In this specification, a method and system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs) is presented, in particular by identifying TCR CDR3 coding nucleic acid sequence clones that constitute T-cell receptor (TCR) complementarity-determining region 3 (CDR3) clonal diversity, thereby facilitating the identification of clinically effective populations of tumor-infiltrating lymphocytes (TILs). [Overview of the project] [Means for solving the problem]

[0005] Summary of the present invention

[0005] The present invention relates to a method and system for identifying clinically effective populations of tumor-infiltrating lymphocytes using various nucleic acid sequence-based profiles of lymphocyte clonal diversity. The present invention is illustrated in numerous embodiments and applications, some of which are summarized below and throughout this specification.

[0006]

[0006] In one embodiment, the present invention is a method for identifying a clinically effective population of tumor-infiltrating lymphocytes (TILs), comprising identifying a clinically effective population of tumor-infiltrating lymphocytes (TILs) in a TIL treatment population administered to a subject, (i) Identify TCR CDR3 coding nucleic acid sequence clones that constitute T cell receptor (TCR) complementarity-determining region 3 (CDR3) clonal diversity in the TIL therapeutic population; (ii) Identify the TCR CDR3 coding nucleic acid sequence clones that constitute the TCR CD3 clonal diversity of the first population of peripheral blood mononuclear cells (PBMCs) isolated from the subjects at least 14 days after administration of the therapeutic population of step (i) to the subjects; (iii) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (ii), determine the frequency of such unique TCR CDR3 clones in the therapeutic population of TIL and the first population of PBMCs, respectively; (iv) Sorting the unique TCR CDR3-encoding nucleic acid sequence clones identified in step (ii) from the maximum frequency to the minimum frequency for each of the treatment population of TILs and the first population of PBMCs; and (v) Selecting, from the first population of PBMCs sorted in step (iv), ten unique TCR CDR3-encoding nucleic acid sequence clones of the highest frequencies, such that TILs expressing such clones in the treatment population of TILs constitute a clinically effective population of TILs, thereby identifying a clinically effective population of TILs A method comprising the above is targeted.

[0007]

[0007] In another aspect, the present invention is a method for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3)-encoding nucleic acid sequence clones in tumor infiltrating lymphocytes (TILs) in a treatment population of TILs administered to a subject, comprising: (a) Identifying TCR CDR3-encoding nucleic acid clones that constitute the TCR CDR3 clone diversity of the treatment population of TILs; (b) Identifying TCR CDR3-encoding nucleic acid sequence clones that constitute the TCR CDR3 clone diversity of a first population of peripheral blood mononuclear cells (PBMCs) isolated from the subject at least 14 days after administering the treatment population of step (a) to the subject; (c) Determining the frequency of such unique TCR CDR3-encoding nucleic acid sequence clones in each of the treatment population of TILs and the first population of PBMCs for each of the unique TCR CDR3-encoding nucleic acid sequence clones identified in step (b); and (d) Comparing the frequency of the TCR CDR3-encoding nucleic acid sequence clones in the first population of PBMCs with the frequency of the TCR CDR3-encoding nucleic acid sequence clones in the treatment population of TILs for each of the unique TCR CDR3-encoding nucleic acid sequence clones identified in step (b) to determine the persistence and activity of the TCR CDR3-encoding nucleic acid sequence clones in the treatment TIL population administered to the subject The method including

[0008]

[0008] In another aspect, the present invention is a system for identifying a clinically effective population of tumor infiltrating lymphocytes (TILs), including a memory; one or more processors; and one or more modules stored in the memory and configured for execution by the one or more processors, the modules being (a) for identifying T cell receptor (TCR) complementarity determining region 3 (CDR3) - encoding nucleic acid sequence clones that constitute the clonal diversity of the treatment population of TILs; (b) for identifying TCR complementarity determining region 3 (CDR3) - encoding nucleic acid sequence clones that constitute the clonal diversity of a first population of peripheral blood mononuclear cells (PBMCs) isolated at least 14 days after administering the treatment population of step (a) to the subject; (c) for determining the frequency of such unique TCR CDR3 clones in each of the treatment population of TILs and the first population of PBMCs for each unique TCR CDR3 - encoding nucleic acid sequence clone identified in step (b); (d) for sorting the unique TCR CDR3 - encoding nucleic acid sequence clones identified in step (b) from the maximum frequency to the minimum frequency for each of the treatment population of TILs and the first population of PBMCs; and (e) selecting, from the first population of PBMCs sorted in step (d), ten unique TCR CDR3 - encoding nucleic acid sequence clones with the maximum frequencies, such that TILs expressing such clones in the treatment population of TILs constitute a clinically effective sub - population of TILs, thereby identifying a clinically effective population of TILs The system including the instructions

[0009]

[0009] In further embodiments of the above - mentioned method and system, DNA, RNA, or both DNA and RNA are used to determine the CDR3 clonal diversity of the treatment population of TILs or the CDR3 clonal diversity of PBMCs.

[0010]

[0010] In another embodiment, the present invention is a method for determining TIL production process equivalence using TCR repertoire analysis, To determine a first set of unique CDR3 sequences expressed by TIL in the first sample; To determine a second set of unique CDR3 sequences expressed by TIL in the second sample; (i) the ratio of the number of unique CDR3 sequences occurring in both the first and second sets to the number of unique CDR3 sequences in the first set, and / or (ii) the ratio of the number of unique CDR3 sequences occurring in both the first and second sets to the number of unique CDR3 sequences in the second set; and Based on the aforementioned ratio, the equivalence of TIL in the first sample and TIL in the second sample is determined. This applies to methods that include [specific methods].

[0011]

[0011] In a further embodiment of the above method, TIL of a first sample is produced in a first facility, and TIL of a second sample is produced in a second facility.

[0012]

[0012] In a further embodiment of the above method, the number of unique CDR3 sequences in the first set correlates with the therapeutic efficacy of TIL in the first sample.

[0013]

[0013] In a further embodiment of the above method, the number of unique CDR3 sequences in the second set correlates with the therapeutic efficacy of TIL in the second sample.

[0014]

[0014] In a further embodiment of the above method, the first sample and the second sample are derived from the same sample.

[0015]

[0015] In a further embodiment of the above method, at least one of the first sample and the second sample is obtained from a subject having a solid tumor.

[0016]

[0016] In a further embodiment of the above method, the solid tumor carcinoma is selected from the group consisting of melanoma (e.g., uveal melanoma), ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, basal cell carcinoma, cancer caused by human papillomavirus, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), brain cancer, glioblastoma (e.g., GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In a further embodiment of the above method, the solid tumor carcinoma is melanoma. In a further embodiment of the above method, the solid tumor carcinoma is cervical cancer.

[0017]

[0017] In a further embodiment of the above method, the TIL in the first sample and the TIL in the second sample are post-rapid expansion process (REP) TIL.

[0018]

[0018] In a further embodiment of the above method, a ratio having a value of about 0.4 or more, about 0.42 or more, about 0.44 or more, about 0.46 or more, about 0.48 or more, about 0.50 or more, about 0.52 or more, about 0.54 or more, about 0.56 or more, about 0.58 or more, or about 0.60 or more indicates equivalence between the first sample and the second sample.

[0019]

[0019] In a further embodiment of the above method, the CDR3 clonal diversity of TIL in the first sample and / or the second sample is determined by DNA sequencing. In a further embodiment of the above method, the CDR3 clonal diversity of TIL in the first sample and / or the second sample is determined by RNA sequencing.

[0020]

[0020] In a further embodiment of the above method, the first set of unique CDR3 sequences consists of a given number of CDR3 sequences expressed at maximum frequency by TIL in the first sample. In a further embodiment of the above method, the second set of unique CDR3 sequences consists of a given number of CDR3 sequences expressed at maximum frequency by TIL in the second sample.

[0021]

[0021] In a further embodiment of the above method, the given number is selected from the group consisting of about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, and more than about 500.

[0022]

[0022] In a further embodiment of the above method, the given number correlates with more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 100% of the total number of sequences in one or both of the first set of unique CDR3 sequences and the second set of unique CDR3 sequences.

[0023]

[0023] In a further embodiment of the above method, the given number is about 10 to about 20. In a further embodiment of the above method, the given number correlates with about 40% of the total number of sequences in one or both of the first set of unique CDR3 sequences and the second set of unique CDR3 sequences. In a further embodiment of the above method, the given number is about 300 to about 400. In a further embodiment of the above method, the given number correlates with about 80% of the total number of sequences in one or both of the first set of unique CDR3 sequences and the second set of unique CDR3 sequences.

[0024]

[0024] These embodiments characterized above, and other embodiments of the present invention, are illustrated in numerous described embodiments and uses, some of which are shown in the drawings and examples and characterized in the following sections of claims. However, the above summary does not describe each of the described embodiments or any of the embodiments of the present invention.

[0025] Brief explanation of the drawing

[0025] The above summary of the present invention and the following detailed description will be better understood when read carefully together with the accompanying drawings. [Brief explanation of the drawing]

[0026] [Figure 1]

[0026] An overview of Example 1 will be given. [Figure 2]

[0027] This paper describes the unique Shannon entropy of CDR3 (uCDR3) chronotypes in patients who respond to TIL therapy and those who do not. [Figure 3]

[0028] This document describes a data table showing clonal persistence determined by the method of the present invention 42 days after administration of the TIL population to clinical trial patients. [Figure 4]

[0029] This paper describes the clonal diversity between responding and non-responding patient groups, as well as the persistence of uCDR3 clones from the administered TIL population in patient PBMCs 42 days after infusion. [Figure 5]

[0030] This explains that the shared uCDR3 clone originates from the administered TIL product. [Figure 6]

[0031] This section describes the plots of persistent clones for each subject derived from administered TIL products. [Figure 7]

[0032] This explains the uCDR3 clonal diversity present in seven successful patients. [Figure 8]

[0033] A summary of the results from Example 1 will be provided. [Figure 9]

[0034] The logic flowchart for uCDR3 chronotype analysis using the method of the present invention is described. [Figure 10]

[0035] This document describes a 22-day process for harvesting, expanding, and preparing infusion TIL products from non-selective polyclonal autologous T cells. [Figure 11]

[0036] Figure 11 illustrates the unique CDR3 sequences of TIL for each response in Cohort 2. Figure 11B illustrates the TIL Shannon entropy (exponent) for each response in Cohort 2. [Figure 12]

[0037] Figure 12A illustrates the number of TIL clones in the TIL product compared to a matching sample of PBMC on day 42. Figure 12B illustrates the percentage of TIL clones in TIL collection (100%), day 42 (55.33%), and pre-infusion circulation (15.13%). [Figure 13]

[0038] This section describes the plots of persistent clones for each subject derived from administered TIL products. Each point represents the rank as a percentage of the TIL product for each of the top-ranking clones. The most frequent clone in the TIL product corresponds to the minimum value; similarly, the least frequent clone in the TIL product corresponds to a value closer to 100. Persistent clones were identified at both high and low levels in the TIL product. uCDR3 clones represented at either high or low frequency in the TIL product could persist for at least 6 weeks after infusion. [Figure 14]

[0039] This section describes the percentage of shared uDCR3 in TILs for each response in Cohort 2. A slight correlation is shown between matched TIL products and D42 PMBC samples. The percentage of persistent clones was determined by dividing the number of clones detected in both TIL products and D42 PMBC samples by the number of unique CDR3 clones in the TIL products, providing a measure of overlap between the composition of infusion products and in vivo circulating T cells. The results are shown as box plots. [Figure 15]

[0040] Figure 15A illustrates the CDR3 across all patients tested. Each white line represents an individual clone, stacked in the upper column for each subject and sorted in descending order based on the number of subjects containing the clone. Figure 15B is a table showing the CDR3 sequences found in more than four patients. It shows the number of subjects with a common clone, the number of clones in each of those groups, and the number of non-tumor-associated clones. [Figure 16]

[0041] This section explains the diagram illustrating the equivalence of manufacturing and testing facilities. [Figure 17A]

[0042] This paper summarizes the stage 3 equivalence of clinical sample iREP data and site-specific sample iREP data obtained using commercially available iRepertoire technology (Huntsville, AL), and explains, for example, the proportion of unique CDR3 sequences shared among the compared samples. [Figure 17B]

[0042] A summary of the stage 3 equivalence of clinical sample iREP data and site-specific sample iREP data obtained using commercially available iRepertoire technology (Huntsville, AL) is presented, as an example, illustrating the proportion of unique CDR3 sequences shared among the samples being compared. [Figure 17C]

[0042] A summary of the stage 3 equivalence of clinical sample iREP data and site-specific sample iREP data obtained using commercially available iRepertoire technology (Huntsville, AL) is presented, as an example, illustrating the proportion of unique CDR3 sequences shared among the samples being compared. [Figure 18]

[0043] This paper summarizes the equivalence of peripheral blood lymphocyte iREP data obtained using commercially available iRepertoire technology (Huntsville, AL), and, as an example, describes the proportion of unique CDR3 sequences shared among the samples being compared. [Figure 19]

[0044] This paper summarizes the Stage 1 equivalence of site-specific sample iREP data obtained using commercially available iRepertoire technology (Huntsville, AL), and, as an example, describes the proportion of unique CDR3 sequences shared among the samples being compared. [Figure 20]

[0045] This paper summarizes the equivalence of iREP data from unrelated tumor-infiltrating lymphocyte samples obtained using commercially available iRepertoire technology (Huntsville, AL), and, as an example, describes the proportion of unique CDR3 sequences shared among the samples being compared. [Figure 21]

[0046] This document summarizes facility-specific sample iREP data obtained from patients with chronic lymphocytic leukemia using commercially available iRepertoire technology (Huntsville, AL). [Figure 22]

[0047] This paper summarizes the equivalence of unrelated peripheral blood lymphocyte sample iREP data obtained using commercially available iRepertoire technology (Huntsville, AL), and, as an example, describes the proportion of unique CDR3 sequences shared among the samples being compared. [Figure 23]

[0048] This provides a table of CDR3 sequences sorted by the proportion of each clone that appears. [Modes for carrying out the invention]

[0027] Detailed description of the present invention

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. All patents and publications referenced herein are incorporated by reference in their entirety.

[0028] definition

[0050] The term "immune repertoire" refers to a set of distinct CDR3 sequences that are detected in lymphocytes of one or more individuals, as appropriate.

[0029]

[0051] The chronotype of the immune repertoire, also known as the "clonal type," is determined by the rearrangement of variable (V), diversity (D), and binding (J) gene segments via somatic recombination during the early stages of immunoglobulin (Ig) and T cell receptor (TCR) production in the immune system. V(D)J rearrangements can be amplified and detected from the alpha, beta, gamma, and delta chains of T cell receptors, as well as from the heavy (IgH) and light (IgK, IgL) chains of immunoglobulins. Cells can be obtained from patients, for example, by obtaining tissue or fluid from peripheral blood, lymphoid tissue, cancerous tissue, or other organs and / or organ systems. Techniques for obtaining these samples, for example, blood samples, are known to those skilled in the art. Cell counts can be estimated from the number of sequences detected by PCR amplification and sequencing.

[0030]

[0052] The CDR3 region, containing approximately 30-90 nucleotides, encapsulates the junction of the gene's recombination variable (V), diversity (D), and binding (J) segments. It encodes receptor binding specificity and is useful as a sequence tag for identifying unique V(D)J rearrangements.

[0031]

[0053] Wang et al. disclosed that PCR can be used to obtain a quantitative or semi-quantitative assessment of the number of target molecules in a sample (Wang, M. et al, “Quantitation of mRNA by the polymerase chain reaction,” Proc. Nat'l. Acad. Sci. 86:9717-9721 (1989)). Methods particularly effective for achieving quantitative amplification have already been described by the present inventors. One such method is known as arm-PCR, which is described in U.S. Patent Application Publication No. 2009 / 0253183A1.

[0032]

[0054] The term "in vivo" refers to events that occur within the subject's body.

[0033]

[0055] The term "in vitro" refers to an event that occurs outside the body of the subject. In vitro assays include cell-based assays that use live or dead cells, as well as cell-free assays that do not use intact cells.

[0034]

[0056] The term "ex vivo" refers to an event that involves processing or performing a procedure on cells, tissues, and / or organs removed from a subject's body. Appropriately, the cells, tissues, and / or organs can be returned to the subject's body by surgical or procedural means.

[0035]

[0057] In this specification, “tumor-infiltrating lymphocytes” or “TILs” refers to the initial population of cells obtained as leukocytes that have left the bloodstream and migrated into the tumor. TILs include, but are not limited to, CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + Examples include T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs can be either primary or secondary. "Primary TILs" are obtained from patient tissue samples (sometimes referred to as "freshly obtained" or "freshly isolated") as outlined herein, while "secondary TILs" are any expanded or proliferated TIL cell populations, as discussed herein, including but not limited to bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). Genetically modified TILs can be considered as TIL cell populations.

[0036]

[0058] In this specification, “population of cells” (e.g., TIL) means a large number of cells that share common traits. Generally, a population is typically 1 × 10⁻⁶ 6 ~1 × 10 10 This is a range of numbers, and different TIL populations contain different numbers. For example, the initial growth of primary TIL in the presence of IL-2 is approximately 1 × 10⁻¹⁶. 8 This results in a population of bulk TILs from individual cells. REP expansion is generally 1.5 × 10⁶ for infusion. 9 ~1.5×1010 performed to provide a population of cells. In some embodiments, the REP expansion is 2.3×10 10 ~13.7×10 10 to provide a population.

[0037]

[0059] As used herein, "cryopreserved TIL" means that the TIL is processed and stored in the range of about -150°C to -60°C, either in primary, bulk, or expanded (REP TIL). General methods of cryopreservation are described elsewhere herein, by way of example, in the Examples. For clarity, "cryopreserved TIL" is distinguishable from frozen tissue samples that can be used as a source of primary TIL.

[0038]

[0060] As used herein, "thawed cryopreserved TIL" means a population of TIL that has been cryopreserved previously and then processed to be returned to a temperature above room temperature, by way of example, but not limited to, cell culture temperature or a temperature at which the TIL can be administered to a patient.

[0039]

[0061] TIL can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve treatment. TIL can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and / or, TIL can be defined functionally by their ability to infiltrate solid tumors upon reintroduction into a patient.

[0040]

[0062] The term "central memory T cells" is CD45R0+ in humans and CCR7 (CCR7 hi ) and CD62L (CD62 hiThis refers to a subset of T cells that constitutively express IL-2. Surface phenotypes of central memory T cells include TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. After TCR induction, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells are dominant in the CD4 compartment of the blood and are relatively high in the lymph nodes and tonsils in humans.

[0041]

[0063] The term "effector memory T cell" is similar to central memory T cells in that it is CD45R0+, but it has lost constitutive expression of CCR7 (CCR7 lo ), CD62L expression is heterogeneous or low (CD62L lo This refers to a subset of human or mammalian T cells. Surface phenotypes of central memory T cells include TCR, CD3, CD127 (IL-7R), and IL-15R. BLIMP1 is a transcription factor for central memory T cells. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, such as interferon-γ, IL-4, and IL-5, after antigenic stimulation. Effector memory T cells are dominant in the CD8 compartment of the blood and are relatively high in the lungs, liver, and intestines in humans. CD8+ effector memory T cells carry large amounts of perforin.

[0042]

[0064] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, such as lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells.

[0043]

[0065] The terms “peripheral blood lymphocytes” and “PBL” refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of T cell phenotype, e.g., CD3+CD45+ T cell phenotype.

[0044]

[0066] As used herein, the term “therapeutic effect” encompasses therapeutic benefits and / or preventive benefits. Preventive effects include delaying or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or improving the progression of a disease or condition, or any combination thereof.

[0045]

[0067] As used herein, the term “clinically effective” with respect to TILs or other T cell populations encompasses clinically detectable therapeutic and / or prophylactic effects. Non-limiting examples of clinically detectable therapeutic effects include reduction of solid tumor masses and reduction of patient-reported symptoms, such as pain or discomfort. Prophylactic effects include delaying or eliminating the onset of disease or condition, delaying or eliminating the onset of symptoms of disease or condition, slowing, halting, or improving the progression of disease or condition, or any combination thereof.

[0046]

[0068] For example, where a range is used in this specification to describe a physical or chemical property, such as molecular weight or chemical formula, it shall include all combinations of the range and its specific embodiments and components of those combinations. The use of the term “about” means that, when referring to a number or numerical range, the number or numerical range referred to is an approximation within experimental variability (or aggregate experimental error), and therefore the number or numerical range may vary. The variation is typically 0% to 15%, preferably 0% to 10%, and more preferably 0% to 5% of the described number or numerical range. The term “comprising” (and related terms, e.g., “comprise” or “comprises” or “having” or “including”) includes their embodiments, e.g., embodiments of any configuration of a substance, method or process that “consists of” or “essentially consists of” the described features.

[0047]

[0069] The compounds of the present invention also include antibodies. The terms “antibody” and its plural form “antibodies” refer to whole immunoglobulins and any antigen-binding fragments ("antigen-binding regions") or single chains thereof. “Antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, or further to its antigen-binding regions. Each heavy chain has a heavy chain variable region (V in this specification). H It includes a heavy chain constant region (abbreviated as CH1, CH2, and CH3). Each light chain has a light chain variable region (V in this specification). L It includes the (abbreviated as) and light chain steady region. The light chain steady region consists of one domain, C L Includes the V antibody. H and V L The region is called the Complementarity Determination Region (CDR) or the Hypervariability Region (HVR), and can be further subdivided into the Hypervariability Region, where more conserved regions called the Framework Region (FR) may be scattered. H and V LIt consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0048]

[0070] The terms “monoclonal antibody,” “mAb,” “monoclonal antibody composition,” or their plural forms, refer to preparations of antibody molecules with a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. The DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of that monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. After isolation, the DNA can be loaded into an expression vector, which is then transfused into host cells, such as Escherichia coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not inherently produce immunoglobulin proteins, thereby achieving the synthesis of monoclonal antibodies in recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0049]

[0071] As used herein, the terms “antigen-binding moiety” or “antigen-binding fragment” (or simply “antibody moiety” or “fragment”) of an antibody refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Examples of binding fragments that fall within the scope of the term “antigen-binding moiety” of an antibody include (i)V L , V H , C L(ii) a monovalent fragment consisting of the CH1 domain, the Fab fragment; (ii) a bivalent fragment F(ab')2 fragment containing two Fab fragments linked by disulfide bridges in the hinge region; (iii) V H and Fd fragment consisting of the CH1 domain; (iv) V of a single arm of the antibody L and V H Fv fragment consisting of domains, (v)V H or V L Examples include domain antibody (dAb) fragments that can consist of domains (Ward, et al., Nature, 1989, 341, 544-546); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, the two domains of the Fv fragment, V L and V H Although encoded by separate genes, they are linked together using a synthetic linker via a recombination method to form V L and V H These can be prepared as single protein chains in which regions pair up to form monovalent molecules known as single-chain Fv (scFv); see, for example, Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also included within the scope of the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same way as intact antibodies.

[0050]

[0072] As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-directed mutagenesis or in vivo by somatic mutation). As used herein, the term "human antibody" does not include antibodies in which CDR sequences derived from other mammalian species, such as the germline of mice, are grafted onto a human framework sequence.

[0051]

[0073] The term "human monoclonal antibody" refers to an antibody exhibiting single-binding specificity, in which both the framework and CDR regions have variable regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by B cells obtained from a transgenic non-human animal having a genome containing human heavy-chain and light-chain trans genes fused to a hybridoma, e.g., an immortalized cell, such as a transgenic mouse.

[0052]

[0074] As used herein, the term “recombinant human antibody” includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, for example, (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) or hybridomas prepared therefrom (as further described below) with respect to the human immunoglobulin gene, (b) antibodies isolated from host cells transformed to express human antibodies, for example, transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of the human immunoglobulin gene sequence to another DNA sequence. Such recombinant human antibodies have variable regions in which the framework and CDR region are derived from the human germline immunoglobulin sequence. However, in some embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if a transgenic animal with respect to the human Ig sequence is used), and thus the V of recombinant antibodies H and V L The amino acid sequence of the region is human germline V H and V L This sequence is derived from and related to other sequences, yet it is a sequence that cannot naturally exist in the in vivo human antibody germline repertoire.

[0053]

[0075] As used herein, "isotype" refers to an antibody class (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. In mammals, there are five antibody isotypes: IgA, IgD, IgG, IgM, and IgE. In humans, there are four subclasses of the IgG isotype: IgG1, IgG2, IgG3, and IgG4, and two subclasses of the IgA isotype: IgA1 and IgA2.

[0054]

[0076] In this specification, the terms "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are used synonymously with the term "antibody that specifically binds to an antigen."

[0055]

[0077] The term "human antibody derivative" refers to any modified form of a human antibody, for example, a conjugate of that antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody-drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated with a therapeutic moiety, for example, a bacterial toxin, a cytotoxic agent, or a radionuclide-containing toxin. The toxic moiety can be conjugated to the antibody of the present invention using methods available in the art.

[0056]

[0078] The terms "humanized antibody," "humanized antibodies," and "humanization" refer to antibodies in which a CDR sequence derived from the germline of another mammalian species, e.g., mouse, is grafted onto a human framework sequence. Additional framework region modifications can be made within the human framework sequence. Humanized non-human (e.g., mouse) antibodies are chimeric antibodies containing a minimal sequence derived from non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced by residues from the 15 hypervariable regions of a non-human species (donor antibody) with desired specificity, affinity, and capability, e.g., mouse, rat, rabbit, or non-human primate. In some examples, Fv framework region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies correspond to non-human immunoglobulins where all or substantially all of the hypervariable loop is a human immunoglobulin sequence, and include at least one, typically two, variable domains where all or substantially all of the FR region is a human immunoglobulin sequence. Humanized antibodies also optionally include the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin Fc. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596.

[0057]

[0079] The term "chimeric antibody" refers to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species; for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody.

[0058]

[0080] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragment has a heavy chain variable domain (V H ) is a light chain variable domain (V L ) linked in the same polypeptide chain (V H -V L or V L -V H ) includes. By using a linker short enough not to allow pairing between two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are described in more detail, for example, European Patent No. 404,097, International Publication No. 93 / 11161; and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0059]

[0081] The term "glycosylation" refers to modified derivatives of antibodies. Aglycoslated antibodies lack glycosylation. Glycosylation can be modified, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Nonglycosylation can increase the affinity of an antibody for an antigen, as described in U.S. Patents 5,714,350 and 6,350,861. Furthermore, antibodies with modified types of glycosylation can be produced, for example, low-fucosylated antibodies with a reduced amount of fucosyl residues or antibodies with an increased bibranched GlcNac structure. Such modified glycosylation patterns have been demonstrated to increase the antibody's capabilities. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in host cells with a modified glycosylation mechanism. Cells possessing altered glycosylation mechanisms have been described in the art and can be used as host cells expressing the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6)fucosyltransferase), and as a result, antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709FUT8- / - cell lines were created by targeted disruption of the FUT8 gene in CHO / DG44 cells using two substitution vectors (see, for example, U.S. Patent Application Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. 1,176,195 describes a cell line in which the function of the FUT8 gene encoding fucosyltransferase is disrupted, and an antibody expressed in such a cell line exhibits low fucosylation due to reduction or elimination of alpha-1,6 linkage-related enzymes, and also describes a cell line that has low or no enzymatic activity for the addition of fucose to N-acetylglucosamine bound to the Fc region of the antibody, such as the rat myeloma cell line YB2 / 0 (ATCC CRL1662). International Publication 03 / 035835 describes Lec13 cells, a mutant CHO cell line exhibiting reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in decreased fucosylation of antibodies expressed in its host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Publication 99 / 54342 describes a cell line engineered to express glycoprotein-modified glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), in which antibodies expressed in the engineered cell line show increased bifurcated GlcNac structure, leading to increased ADCC activity of the antibodies (Umana, et al.). (See also al., Nat. Biotech. 1999, 17, 176-180). Alternatively, the fucose residues of antibodies can be cleaved using a fucosidase enzyme. For example, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523, fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies.

[0060]

[0082] "PEGylation" refers to a modified antibody or antibody fragment that is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that one or more PEG groups adhere to the antibody or antibody fragment. Pegylation can, for example, increase the biological (e.g., serum) half-life of an antibody. Preferably, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to any form of PEG used to derivatize other proteins, e.g., mono(C1-C1) 10 ) encompasses alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated may be a non-glycosylated antibody. Pegylation methods are known in the art, as described, for example, in European Patent Nos. 0154316 and 0401384, and can be applied to the antibody of the present invention.

[0061]

[0083] The term "biosimilar" refers to a biological product that is highly similar to a brand-name biological product approved in the United States, despite minor differences in clinically inactive components, and to which there is no clinically significant difference between the biological product and the brand-name product in terms of safety, purity, and efficacy. Furthermore, a generic or "biosimilar" drug is a biological drug that is similar to another biological drug already approved for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory bodies in other countries and regions. A biological product or biological drug is a drug made from or derived from a biological resource, such as bacteria or yeast. These may consist of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the original anti-CD20 monoclonal antibody is rituximab, then an anti-CD20 biosimilar monoclonal antibody approved by the regulatory authority in accordance with rituximab is a "biosimilar" to rituximab, or "its biosimilar" to rituximab. In Europe, a generic or "biosimilar" drug is a biological drug that is similar to another biological drug already authorized for use by the European Medicines Agency (EMA). The relevant legal basis for generic applications in Europe is Article 6 of Regulation (EC) 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC (or its amended version, if any), and therefore in Europe, biosimilars may be authorized, approved for authorization, or subject to application for authorization under Article 6 of Regulation (EC) 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. Original biopharmaceutical formulations that have already been approved may be referred to as "original formulations" in Europe. Some of the requirements for a formulation to be considered a biosimilar are outlined in the CHMP Guideline on Similar Biological Medicinal Products. In addition, formulation-specific guidelines, including those for monoclonal antibody biosimilars, are provided by the EMA for each formulation and are available on its website.The biosimilars described herein may be similar to the original drug product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. In addition, the biosimilars may be used to treat, or intended to treat, the same medical conditions as the original drug product. Therefore, the biosimilars described herein can be considered to have quality characteristics similar to or highly similar to the original drug product. Alternatively, or in addition, the biosimilars described herein can be considered to have biological activity similar to or highly similar to the original drug product. Alternatively, or in addition, the biosimilars described herein can be considered to have a safety profile similar to or highly similar to the original drug product. Alternatively, or in addition, the biosimilars described herein can be considered to have efficacy similar to or highly similar to the original drug product. As described herein, biosimilars in Europe are compared to original drug products approved by the EMA. However, in some cases, biosimilars can be compared to biopharmaceutical formulations approved in trials outside the European Economic Area (non-EEA approved “control drug”). Such trials include, for example, certain clinical and in vivo non-clinical trials. The term “biosimilar” as used herein also refers to biopharmaceutical formulations that have been compared to, or can be compared to, a non-EEA approved control drug. Certain biosimilars are proteins, e.g., antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor amino acid structural modifications (e.g., including, for example, amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. Biosimilars may contain amino acid sequences with 97% or more, e.g., 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of their original drug formulation. A biosimilar may have one or more post-translational modifications that differ from those of the original drug formulation, such as, but not limited to, glycosylation, oxidation, deamidation, and / or truncation, provided that the difference does not result in any change in the safety and / or efficacy of the drug formulation.Biosimilars may have the same or different glycosylation patterns as the original drug formulation. In particular, but not limited to, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the original drug formulation. In addition, biosimilars may deviate from the original drug formulation, for example, in terms of their potency, drug form, formulation, excipients, and / or delivery method, provided that the safety and efficacy of the drug formulation are not compromised. Biosimilars may include differences compared to the original drug formulation, for example, in terms of their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles, but are still considered sufficiently similar to the original drug formulation to be considered approved or suitable for approval. In certain circumstances, a biosimilar may exhibit different binding properties compared to the original drug formulation, and these different binding properties are not considered to preclude approval as a generic biopharmaceutical by regulatory authorities, such as the EMA. The term “biosimilar” is also used synonymously by regulatory bodies in other countries and regions.

[0062]

[0084] The terms “sequence identity,” “percent identity,” and “sequence percent identity” relating to two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are identical, or have a specified percentage of identical nucleotide or amino acid residues, when compared to maximize correspondence and aligned (with gaps introduced as necessary), without considering any conserved amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. A suitable program for determining percent sequence identity is, for example, the BLAST program suite available from the U.S. government’s National Center for Biotechnology Information BLAST website. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Alignments can be generated using ClustalW and ClustalX, see Larkin et al., Bioinformatics 23:2947-2948 (2007); Goujon et al., Nucleic Acids Research, 38 Suppl:W 695-9 (2010); and McWilliam et al., Nucleic Acids Research 41 (Web Server issue):W 597-600 (2013). Those skilled in the art can determine appropriate parameters for maximum alignment using specific alignment software.In one embodiment, default parameters of the alignment software are used.

[0063]

[0085] The transitional clauses “contains,” “essentially consists of,” and “consist of,” when used in the claims attached to the original and amended forms of the application, define the scope of the claims in terms of what is excluded from the claims if there are additional elements or steps of the claims that are not described. The term “contains” is intended to be inclusive or open-ended and does not exclude any additional undescribed elements, methods, steps, or materials. The term “consist of” excludes any elements, steps, or materials other than those specified in the claim, and in the latter example, also excludes impurities that normally accompany the specified material. The term “essentially consists of” limits the scope of the claim to those that do not substantially affect the specified elements, steps, or materials and the basic and novel features of the claimed invention. All compositions, methods, and kits described herein that embody the present invention may, in alternative embodiments, be more specifically defined by any of the transitional clauses “contains,” “essentially consists of,” and “consist of.”

[0064]

[0086] The term "solid tumor" refers to an abnormal mass of tissue that does not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. The term "solid tumor carcinoma" refers to a malignant, neoplastic, or cancerous solid tumor. Examples of solid tumor carcinomas include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. In some embodiments, cancer is selected from cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The tissue structure of a solid tumor includes interdependent tissue compartments comprising parenchyma (cancer cells) and supporting stromal cells that can disperse the cancer cells and provide a supporting microenvironment.

[0065]

[0087] The term "hematological malignancies" refers to cancers and tumors of hematopoietic and lymphoid tissues in mammals, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also called "humoral malignancies." Examples of hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, and non-Hodgkin lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies that affect B cells.

[0066]

[0088] As used herein, the terms “equivalent” or “equivalence” may refer to two or more samples having similar qualities. In one embodiment, two or more equivalent samples have the same or nearly the same therapeutic efficacy. In one embodiment, two or more “equivalent” samples have the same or nearly the same CDR3 clonal diversity. The use of the term “about” indicates an approximation within experimental variability (or statistical experimental error). Variability is typically 0% to 15%, preferably 0% to 10%, and more preferably 0% to 5% of the number or numerical range described.

[0067]

[0089] To avoid misunderstanding, it is intended that any specific features (e.g., integers, characteristics, values, uses, diseases, formulas, compounds, or groups) described herein in conjunction with any particular aspect, embodiment, or example of the present invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless otherwise incompatible. Thus, such features may, where appropriate, be used in conjunction with any of the definitions, claims, or embodiments defined herein. All features disclosed herein (including any appended claims, abstracts, and drawings) and / or all steps of any method or process so as disclosed may be combined in any combination, except for any combination in which at least some of the features and / or steps are mutually exclusive. The present invention is not limited to any detail of any disclosed embodiment. The present invention extends to any novel one or novel combination of any features disclosed herein (including any appended claims, abstracts, and drawings), or any novel one or novel combination of any method or step so as disclosed.

[0068] TIL Enlargement Method

[0090] Various methods for expanding tumor-infiltrating lymphocytes to produce a therapeutic population for TILs are known in the art. The following methods are not limiting. It is understood that the systems and methods disclosed herein are compatible with all methods for expanding, culturing, and producing TILs, bone marrow-infiltrating lymphocytes (MILs), and PBLs.

[0069]

[0091] An exemplary TIL process known as Process 2A is disclosed in U.S. Patent No. 10,130,659, which is incorporated by reference in its entirety, and of course note Figure 2 comparing Process 1C with Process 2A, and various embodiments of Process 2A, as disclosed in U.S. Patent No. 10,130,659. An exemplary Process 2A TIL manufacturing method is disclosed in U.S. Patent Application Publication 2018 / 0282694A1, which is incorporated by reference in its entirety. An exemplary Process 2A TIL manufacturing method is also disclosed in U.S. Patent Application Publication 2018 / 0207201A1, which is incorporated by reference in its entirety.

[0070]

[0092] Other methods for enlarging TILs are discussed below: Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. Rohann et al., Journal for ImmunoTherapy of Cancer 2018, 6: 102-118 discusses common TIL production and the clinical use of therapeutic TIL populations.

[0071]

[0093] TIL can be expanded by any of the above methods to produce a population of cells suitable for use in the methods and systems disclosed herein.

[0072] CDR3 Chronotype Method nucleic acid amplification method

[0094] Whole cellular nucleic acids can be isolated from a population of lymphocytes and amplified using various methods, for example, dimer-avoided multiplex polymerase chain reaction (dam-PCR), one embodiment of multiplex PCR, disclosed in International Publication No. 2018 / 165593 and International Publication PCT / US2018 / 021816. Han et al., and / or U.S. Patent No. 9,938,578, disclose a multiplex method for analyzing a mixed nucleotide sample, comprising producing an amplified product tagged with one or more target sequences by non-interfering, non-cancellable target-specific polynucleotide identification tags using polynucleotide amplification, and pyrosequencing the amplified product via the non-cancellable target-specific polynucleotide identification tag sequences to detect the presence of one or more specific polynucleotide identification tags. The presence of specific polynucleotide identification tags correlates with the presence of specific target sequences.

[0073]

[0095] Other useful methods, though not limited to those disclosed by Han in U.S. Patent Application Publication 2017 / 0088895A1, which is incorporated by reference in whole, focus particularly on methods for amplifying and assembling a representative collection of CDR3 diversity present in a resource sample using primers directed to CDR3 sequences. Such methods include determining the diversity of a patient's immune repertoire by comparing CDR3 sequences in a patient sample with the most commonly shared CDR3 sequences (i.e., pCDR3) of the individual's index group. This proportion of pCDR3 in a patient's sample is referred to as the “normality index” and may serve as a diagnostic indicator of immune repertoire diversity in such a patient. The disclosure further includes methods for determining such a subset of highly shared pCDR3s. In one aspect of the disclosure, a patient's immune repertoire is considered normal if the patient’s normality index matches or exceeds a minimum proportion, while a patient’s immune repertoire is considered abnormal if the patient’s normality index is less than such a minimum proportion.

[0074]

[0096] The CDR3 expressed by individuals exhibits extremely high diversity, up to 10 15 It is conceivable that there are unique CDR3s. Therefore, this disclosure uses CDR3 as a criterion for immune system diversity. Han (U.S. Patent Application Publication 2017 / 0088895A1) discloses that, based on a sampling of 75 million CDR3s, approximately 81% of randomly selected CDR3s are unique to a given individual and are not shared among multiple individuals. Han (U.S. Patent Application Publication 2017 / 0088895A1) provides a method for determining a pool of highly shared CDR3s, thereby enabling a standard index of shared CDR3s that can identify diversity in an individual's immune repertoire. Furthermore, the immune repertoire of a single subject at two different point in time can be determined and then compared as detailed below using the method disclosed herein.

[0075]

[0097] Lymphocytes, such as T cells and / or T cell subsets, can be isolated and / or sorted using techniques known in the art, including, but not limited to, apheresis, separation of PBMCs using Ficoll-paque gradients, FACS, and magnetic bead separation methods.

[0076]

[0098] To determine an immunorepertoire containing a set of distinct CDR3 sequences, the following exemplary approaches can be used: (a) amplifying polynucleotides from a population of leukocytes from a patient in a reaction mix containing target-specific nested primers to produce a first set of amplicons, wherein at least a portion of the target-specific nested primers contains additional nucleotides that serve as templates for the incorporation of a binding site for at least one common primer into the first amplicon during amplification; (b) a portion of the first reaction mix containing the first amplicon into at least one common primer (c) transferring to a second reaction mix containing a lymer; (d) amplifying the first amplicon using at least one common primer to produce a set of second amplicons; (e) sequencing the second amplicons to identify CDR3 sequences in a subpopulation of leukocytes; and (f) quantifying the proportion of pCDR3 represented by the sample using the identified CDR3 sequences to provide a normality index; and (g) identifying whether the normality index is normal or abnormal, with a normal state characterized by the presence of a minimum proportion of pCDR3 and an abnormal state characterized by the absence of a minimum proportion of pCDR3. Such methods are known in the art and are described by Han in U.S. Patent Application Publication No. 2017 / 0088895A1.

[0077]

[0099] Other useful methods are disclosed by Han in U.S. Patent No. 7,999,092, which is incorporated by reference in whole, and arm-PCR and arm-RT-PCR are noted. In summary, Han (U.S. Patent No. 7,999,092) describes a method for amplifying nucleic acids to enable their detection, comprising the steps of: amplifying one or more target nucleic acids using a high concentration of target-specific primers in a first amplification reaction to produce at least one nucleic acid amplicon containing at least one common primer binding site; rescuing at least one nucleic acid amplicon; and amplifying at least one nucleic acid amplicon in a second amplification reaction using a common primer that binds to at least one common primer binding site. One aspect of the present invention utilizes nested target-specific primers. The target nucleic acids may include DNA and / or RNA, and may include human genomic DNA and / or RNA. Amplification can be carried out by polymerase chain reaction (PCR) and / or RT-PCR. The target nucleic acid resource may come from one or more clinical, environmental, or food samples, and the method can be used in various ways, for example, in clinical diagnosis, environmental sampling, plant testing, food safety analysis, detection of genetic disorders, and / or detection of disease pathology. The method can be used in human and / or veterinary medical diagnosis.

[0078]

[0100] Another exemplary approach using the various Han disclosures incorporated herein by reference and above to determine the CDR3 repertoire of lymphocyte populations is arm-PCR and / or arm-RT-PCR in a multi-step reaction to quantitatively amplify the immunorepertoire. During the first round of PCR, nested gene-specific primers targeting the V and C genes, respectively, are used. Forward primers, Fo (forward out) and Fi (forward in), are localized to the V gene. Reverse primers, Ro (reverse out) and Ri (reverse in), are localized to the C gene, respectively. The Fi and Ri primers also include sequencing adapters B and A for the Roche 454 platform (454 and GS Junior), respectively. For the Ri primer, a barcode is also present between sequencing primer A and the C gene-specific primer. As a result, sequencing is limited to single-end reads from primer A only. The second round of PCR is performed using common (sequencing) primers B and A. After gel purification, the obtained product is available for high-throughput sequencing using the Roche 454 platform. No additional enzymatic steps are required. The first round of PCR introduces barcode and sequencing primers into the PCR product. The exponential amplification phase is achieved in the second round of PCR using a common primer; thus the entire repertoire is amplified uniformly and semi-quantitatively without introducing additional amplification bias. Genomic DNA, RNA, mRNA, mixed cell nucleic acid samples, and / or cDNA libraries are suitable starting points for such immunorepertoire amplification methods.

[0079]

[0101] Alternative approaches for measuring and determining chronotype profiles are disclosed in U.S. Patent No. 10,155,992, which is incorporated by reference in whole, and note in particular a method for amplifying the reconstituted T cell receptor CDR3 coding region DNA molecule. Faham and Willis teach a detailed method for developing the T cell receptor (TCR) CDR3 chronotype from the amplified DNA. Such a method complements the RNA focus method and is more advantageously used in the present invention. In one embodiment, the present invention provides a method for identifying clinically effective TIL populations using both mRNA chronotype data and DNA.

[0080]

[0102] Because recombination exists in the DNA of adaptive immune cells in each individual and their associated DNA transcripts, either RNA (e.g., mRNA) or DNA can be sequenced in the method of the invention provided. Recombinant sequences from T cells or B cells encoding T cell receptors or immunoglobulin molecules, or parts thereof, are called chronotypes. DNA or RNA (e.g., mRNA) may correspond to sequences from T cell receptor (TCR) genes or immunoglobulin (Ig) genes encoding antibodies. For example, DNA and RNA may correspond to sequences encoding the α, β, γ, or δ chains of the TCR. In the vast majority of T cells, the TCR is a heterodimer consisting of α and β chains. The TCR α chain is generated by VJ recombination, and the β chain receptor is generated by V(D)J recombination. In humans, the TCR β chain has 48 V segments, 2 D segments, and 13 J segments. Several bases may be deleted and other bases may be added at each of the two junctions (called N and P nucleotides). In a small number of T cells, the TCR consists of γ and δ delta chains. The TCRγ chain is generated by VJ recombination, and the TCRδ chain is generated by V(D)J recombination (Kenneth Murphy et al., Janeway's Immunology 9th edition, Garland Science, 2016, ISBN-13:978-0815345503).

[0081]

[0103] A key feature of the method taught in U.S. Patent No. 10,559,992 is that chronotype expression can be measured at the cellular level. For example, lymphocytes can be counted by measuring chronotypes derived from genomic DNA and the same chronotypes derived from RNA, thereby determining the cell-based expression of the chronotype. A method for simultaneously measuring lymphocyte count and chronotype expression levels in a sample may include: (a) obtaining a sample containing T cells and / or B cells from an individual; (b) sequencing spatially isolated individual molecules derived from the genomic DNA of the cells, wherein such spatially isolated molecules include a number of chronotypes corresponding to the number of lymphocytes in the sample; (c) sequencing spatially isolated individual molecules derived from the RNA of the cells, wherein such spatially isolated individual molecules include a number of chronotypes corresponding to their expression level in the lymphocytes of the sample; and (d) determining the chronotype expression level in the lymphocytes of the sample by comparing, for each chronotype, the number determined from the isolated individual molecules derived from the genomic DNA of the cells and the number determined from the isolated individual molecules derived from the RNA of the cells.

[0082]

[0104] Guidelines for performing multiplex PCR of such immunomolecules can be found in the following references, which are incorporated herein by reference: Morley, U.S. Patent No. 5,296,351; Gorski, U.S. Patent No. 5,837,447; Dau, U.S. Patent No. 6,087,096; Von Dongen et al., U.S. Patent Application Publication No. 2006 / 0234234; European Patent No. 1544308B1, all of which are incorporated herein by reference.

[0083]

[0105] Other means for amplifying nucleic acids that can be used in the methods of the invention provided include, for example, reverse transcription PCR, real-time PCR, quantitative real-time PCR, digital PCR (dPCR), digital emulsion PCR (dcPCR), clone PCR, amplified fragment length polymorphism PCR (AFLP PCR), allele-specific PCR, assembly PCR, asymmetric PCR (using a large excess of primers for the selected strand), colony PCR, helicase-dependent amplification (HDA), hot-start PCR, inverse PCR (IPCR), in situ PCR, long PCR (extension of DNA greater than about 5 kilobases), multiplex PCR, nested PCR (using pairs of two or more primers), single-cell PCR, touchdown PCR, loop-mediated isothermal PCR (LAMP), and nucleic acid sequence-based amplification (NASBA). Other amplification schemes include ligase chain reactions, branched DNA amplification, rolling circle amplification, circle-to-circle amplification, SPIA amplification, target amplification by capture and ligation (TACL) amplification, and RACE amplification.

[0084]

[0106] Information in RNA (e.g., mRNA) in a sample can be converted to cDNA using reverse transcription. Poly-A primers, random primers, and / or gene-specific primers can be used in the reverse transcription reaction according to conventional protocols.

[0085]

[0107] Furthermore, individual nucleic acid molecules can be isolated, optionally re-amplified, and then individually sequenced, for example, by amplification of DNA from the genome (or amplification of nucleic acids in the form of cDNA by reverse transcription of RNA or mRNA). Exemplary amplification protocols can be formed in van Dongen et al., Leukemia, 17:2257-2317 (2003) or van Dongen et al., U.S. Patent No. 8,859,748, which are incorporated by reference in their entirety. In short, an exemplary protocol is as follows: Reaction buffer: ABI Buffer II or ABI Gold Buffer (Life Technologies, San Diego, Calif.); 50 μL final reaction volume; 100 ng sample DNA; 10 pmol each primer (to be adjusted to balance amplification as described below); dNTP at a final concentration of 200 μM; MgCl2 at a final concentration of 1.5 mM (to be optimized according to the target sequence and polymerase); Taq polymerase (1-2 U / tube); Cycling conditions: Pre-activation at 95°C for 7 minutes; Annealing at 60°C; Cycling time: Denaturation for 30 seconds; Annealing for 30 seconds; Extension for 30 seconds.

[0086]

[0108] Methods for isolating nucleic acids from a pool include, but are not limited to, two-dimensional spatial separation of molecules on a solid-phase substrate (e.g., a glass slide), three-dimensional spatial separation of molecules in a solution within a micelle (e.g., this can be achieved using an oil emulsion with or without immobilization of molecules on a solid-phase surface, e.g., on beads), or the use of a microreaction chamber in a microfluidic or nanofluidic chip. Dilution can be used to ensure that, on average, a single molecule is present in a given volume, spatial region, bead, or reaction chamber. Guidelines for such methods of isolating individual nucleic acid molecules can be found in the following references: Green and Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2012, ISBN: 978-936113-42-2); Shendure et al., Science, 309:1728-1732 (including supplementary materials) (2005); U.S. Patent No. 6,300,070; Bentley et al., Nature 456:53-59 (including supplementary materials) (2008); U.S. Patent No. 7,323,305; Matsubara et al., Biosensors & Bioelectronics, 20:1482-1490 (2005); U.S. Patent No. 6,753,147. All of these are incorporated herein by reference.

[0087]

[0109] Furthermore, any high-throughput technique for sequencing nucleic acids can be used in the method of the present invention. DNA sequencing techniques include dideoxysequencing reactions (Sangar method) using labeled terminators or primers and gel separation in slabs or capillaries, sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, allele-specific hybridization of labeled oligonucleotide probes to a library, sequencing by synthesis using allele-specific hybridization and subsequent ligation of labeled clones to a library, real-time monitoring of the incorporation of labeled nucleotides during polymerization steps, polony sequencing, and SOLiD sequencing. Sequencing of isolated molecules has recently been demonstrated by sequential or single extension reactions using polymerases or ligases, and by single or sequential differential hybridization using probe libraries. These reactions have been carried out in parallel for many clone sequences, exemplified by the current commercial applications demonstrating the parallel sequences of over 100 million sequences.

[0088]

[0110] Therefore, these sequencing approaches can be used to test the repertoire of T cell receptors (TCRs) and / or B cell receptors (BCRs). In one embodiment of the present invention, a high-throughput sequencing method is used, which includes the step of spatially isolating individual molecules on a solid-phase surface and sequencing them in parallel on that surface. Such solid-phase surfaces include non-porous surfaces (e.g., those used in Solexa sequencing, e.g., Bentley et al., Nature 456:53-59 (2008) or Complete Genomic sequencing, e.g., Drmanac et al., Science 327:78-81 (2010)), arrays of wells that may contain beads or particle-binding templates (e.g., those using 454, e.g., Margulies et al., Nature 437:376-380 (2005); Ion Torrent sequencing, U.S. Patents 8,574,835), microfabricated films (e.g., those using SMRT sequencing, e.g., Eid et al., Science 323:133-138 (2009)), or bead arrays (e.g., those using SOLiD sequencing or Poloni sequencing, e.g., Kim et al., Science) 316:1481-1414 (2007) can be cited. In various chronotype amplifications, such nucleic acids are amplified in parallel by bridge PCR as described in Bentley et al (cited above) and the manufacturer's instructions (e.g., TruSeq® Sample Preparation Kit and Data Sheet, Illumina, Inc., San Diego, Calif., 2010); and further references: U.S. Patent No. 6,090,592; No. 6,300,070; No. 7,115,400; or European Patent No. 0972081B1, as referenced by reference, to form separate clonal populations or clusters, which are then sequenced.

[0089]

[0111] In one embodiment, the individual molecules that are arranged and amplified on the solid-phase surface are located at 1 cm 2 At least 105 In terms of the density of individual clusters; or 1 cm 2 At least 5 x 10 5 In terms of density of individual units; or 1 cm 2 At least 10 6 Clusters are formed at a density of 1 clusters. In one embodiment, sequencing chemistry with a relatively high error rate is used. In such an embodiment, the average quality score produced by such chemistry is a monotonically decreasing function of the sequence read length. In one embodiment, such a decrease corresponds to 0.5 percent of sequence reads having at least one error at positions 1-75; 1 percent of sequence reads having at least one error at positions 76-100; and 2 percent of sequence reads having at least one error at positions 101-125.

[0090]

[0112] Additional methods useful for determining immune cell chronotypes, particularly for identifying TCR CDR3 coding nucleic acid sequence clones that constitute T cell receptor (TCR) complementarity-determining region 3 (CDR3) clonal diversity in TIL therapeutic populations, are disclosed in U.S. Patents 10,150,996; 10,077,478; 10,077,473; 10,066,265; 9,824,179; 9,528,160; and 9,499,865, each of which is invoked collectively by reference, with particular focus on amplification methods.

[0091]

[0113] Similarly, useful methods for determining immune cell chronotypes, particularly for identifying TCR CDR3 coding nucleic acid sequence clones that constitute T cell receptor (TCR) complementarity-determining region 3 (CDR3) clonal diversity in populations of PBMCs, are disclosed in U.S. Patents 10,150,996; 10,077,478; 10,077,473; 10,066,265; 9,824,179; 9,528,160; and 9,499,865. Such methods may be equivalent to or complement the mRNA-based methods described herein.

[0092] How to identify clinically effective populations for TIL

[0114] In one embodiment, the present invention relates to a method for identifying a clinically effective population of tumor-infiltrating lymphocytes (TILs), comprising identifying a clinically effective population of tumor-infiltrating lymphocytes (TILs) in a TIL treatment population administered to a subject, (i) Identify TCR CDR3 coding nucleic acid sequence clones that constitute T cell receptor (TCR) complementarity-determining region 3 (CDR3) clonal diversity in the TIL therapeutic population; (ii) Identify the TCR CDR3 coding nucleic acid sequence clones that constitute the TCR CD3 clonal diversity of the first population of peripheral blood mononuclear cells (PBMCs) isolated from the subjects at least 14 days after administration of the therapeutic population of step (i) to the subjects; (iii) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (ii), determine the frequency of such unique TCR CDR3 clones in the therapeutic population of TIL and the first population of PBMCs, respectively; (iv) Sort the unique TCR CDR3 coding nucleic acid sequence clones identified in step (ii) from highest to lowest frequency for the TIL treatment population and the first population of PBMCs, respectively; and (v) Select from the first population of PBMCs sorted in step (iv) 10 most frequent unique TCR CDR3 coding nucleic acid sequence clones such that TILs expressing such clones in the TIL therapeutic population constitute a clinically effective population of TIL, thereby identifying a clinically effective population of TIL. This applies to methods that include [specific methods].

[0093]

[0115] In various embodiments, identifying TCR CDR3 coding nucleic acid sequence clones constituting T cell receptor (TCR) complementarity-determining region 3 (CDR3) clonal diversity is carried out using methods known to those skilled in the art, including, but not limited to, the methods described herein. In various embodiments, identifying TCR CDR3 coding nucleic acid sequence clones constituting TCR CDR3 clonal diversity in a first population of peripheral blood mononuclear cells (PBMCs) is carried out using methods known to those skilled in the art, including, including, but not limited to, the methods described herein.

[0094]

[0116] In another embodiment, the present invention relates to a method for identifying a clinically effective population of tumor-infiltrating lymphocytes (TILs), further comprising the steps of (a) identifying TCR CDR3 coding nucleic acid sequence clones constituting the TCR CDR3 clonal diversity of a second population of PBMCs isolated from a subject prior to administration of a therapeutic population of TILs to the subject; and (b) determining the frequency of each unique TCR CDR3 coding nucleic acid sequence clone identified in step (a).

[0095]

[0117] In another embodiment, the present invention relates to a method for identifying a clinically effective population of tumor-infiltrating lymphocytes (TILs), wherein the TCR CDR3 coding nucleic acid sequence clones constituting the clonal diversity of a second population of PBMCs are different from the TCR CDR3 coding nucleic acid sequence clones constituting the clonal diversity of a first population of PBMCs isolated from a subject after administration of a therapeutic population of TILs.

[0096]

[0118] In another embodiment, the present invention relates to a method for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), comprising determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in at least one population by DNA sequencing.

[0097]

[0119] In some embodiments, the present invention relates to a method for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs) by determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in at least one population by RNA sequencing. In some embodiments, the present invention relates to a method for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs) by determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in a therapeutic population of TILs and the frequency of unique TCR CD3 coding nucleic acid sequence clones identified in a first population of PBMCs by both DNA and RNA sequencing.

[0098]

[0120] Another aspect of the present invention provides a method for identifying a clinically effective population of tumor-infiltrating lymphocytes (TILs), comprising comparing the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in a first population of PBMCs determined by RNA sequencing with the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in a first population of PBMCs determined by DNA sequencing, wherein the frequency of such unique clones determined by RNA sequencing indicates a clinically effective population of TILs compared with the frequency of such unique clones determined by DNA sequencing.

[0099]

[0121] Another aspect of the present invention provides a method for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs) in which the frequency of such unique clones is large when determined by RNA sequencing. Another aspect of the present invention provides a method for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs) in which the frequency of such unique clones is large when determined by DNA sequencing.

[0100]

[0122] A further aspect of the present invention provides a method for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs) in which the frequency of such unique clones determined by RNA sequencing correlates with populations of TILs having improved therapeutic efficacy. Another aspect of the present invention provides a method for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs) in which the frequency of such unique clones determined by DNA sequencing does not correlate with populations of TILs having improved therapeutic efficacy. Another aspect of the present invention provides a method for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs) in which the frequency of such unique clones determined by RNA sequencing correlates with populations of TILs having improved therapeutic efficacy, and the frequency of such unique clones determined by DNA sequencing does not correlate with populations of TILs having improved therapeutic efficacy.

[0101]

[0123] In another embodiment, the present invention relates to a method for identifying a clinically effective population of tumor-infiltrating lymphocytes (TILs), wherein the TCR CDR3 coding nucleic acid sequence clone is an mRNA clone identified by RNA sequencing.

[0102]

[0124] The method described herein can be performed at various time points after administration of the therapeutic population of cells. The method for identifying the clinically effective population of TIL is performed at approximately 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 days after administration of the therapeutic population of TIL. It can be implemented approximately 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and / or approximately 60 days later.

[0103]

[0125] In some embodiments, the method for identifying a clinically effective population of TIL is performed approximately 20, 25, 30, 35, 40, 42, 45, 50, 55, and / or 60 days after administration of the TIL treatment population. In other embodiments, the method for identifying a clinically effective population of TIL can be performed approximately 20, 25, 30, 35, 40, 42, 45, 50, 55, 60, 90, 120, 180 days, 1 year, 2 years, 3 years, 4 years, and / or 5 years after administration of the TIL treatment population.

[0104]

[0126] In some embodiments, a method for identifying a clinically effective population for TIL is to administer the TIL treatment population at approximately 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34 days. mRNA can be detected approximately 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and / or 60 days later.

[0105]

[0127] In some embodiments, a method for identifying a clinically effective population of TILs may detect mRNA approximately 20, 25, 30, 35, 40, 42, 45, 50, 55, and / or 60 days after administration of the TIL treatment population. In other embodiments, a method for identifying a clinically effective population of TILs may detect mRNA approximately 20, 25, 30, 35, 40, 42, 45, 50, 55, 60, 90, 120, 180 days, 1 year, 2 years, 3 years, 4 years, and / or 5 years after administration of the TIL treatment population.

[0106]

[0128] Another aspect of the present invention provides a method for improving a target T cell repertoire, comprising (i) identifying a clinically effective population of tumor-infiltrating lymphocytes in accordance with the present disclosure; and (ii) selecting and expanding the population identified in step (i) to produce a second clinically effective therapeutic population for TILs. In another aspect, the present invention further provides administering the expanded cells produced in step (ii) to thereby improve a target T cell repertoire.

[0107]

[0129] In some embodiments, nucleic acids are analyzed from a sample of a subset of cells. For example, methods can be used to separate cells by using cell surface markers. For example, cells can be isolated by cell sorting flow cytometry, flow sorting, fluorescence-activated cell sorting (FACS), bead-based separation, such as magnetic cell sorting (MACS; e.g., using antibody-coated magnetic particles), size-based separation (e.g., sieving or filtering), sorting in a microfluidic apparatus, antibody-based separation, sedimentation, affinity adsorption, affinity extraction, or density gradient centrifugation. Sorting may be based on cell size, morphology, or intracellular or extracellular markers. Methods for isolating or sorting tumor cells are described, for example, in Nagrath et al. Nature 450:1235-1239 (2007); U.S. Patents Nos. 6,008,002, 7,232,653 and 7,332,288; International Publication 2008157220A1; and U.S. Patent Application Publications 2008 / 0138805A1 and 2009 / 0186065; or Rosenberg et al. Cytometry 49:150-158 (2002), each of which is incorporated herein by reference in whole.

[0108] Methods for determining sustainability

[0130] In one embodiment, the present invention provides a method for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic population of TILs administered to a subject, (a) Identify TCR CDR3 coding nucleic acid clones that constitute the TCR CDR3 clonal diversity in the TIL treatment population; (b) Identify the TCR CDR3 coding nucleic acid sequence clones that constitute the TCR CDR3 clonal diversity of a first population of peripheral blood mononuclear cells (PBMCs) isolated from the subjects at least 14 days after administration of the therapeutic population of step (a) to the subjects; (c) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (b), determine the frequency of such unique TCR CDR3 coding nucleic acid sequence clones in the therapeutic population of TIL and the first population of PBMCs, respectively; and (d) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (b), the frequency of the TCR CDR3 coding nucleic acid sequence clone in the first population of PBMCs is compared with the frequency of the TCR CDR3 coding nucleic acid sequence clone in the TIL treatment population to determine the persistence and activity of the TCR CDR3 coding nucleic acid sequence clone in the treatment TIL population administered to the subjects. This applies to methods that include [specific methods].

[0109]

[0131] In another embodiment, the present invention relates to a method for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic population of TILs administered to a subject, wherein the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in a therapeutic population of TILs and the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in a first population of PBMCs is determined by both DNA and RNA (e.g., mRNA) sequencing.

[0110]

[0132] In another embodiment, the present invention relates to a method for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic population of TILs administered to a subject, wherein the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in a first population of PBMCs determined by RNA (e.g., mRNA) sequencing is compared with the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in a first population of PBMCs determined by DNA sequencing, and the frequency of such unique clones determined by RNA (e.g., mRNA) sequencing compared with the frequency of such unique clones determined by DNA sequencing indicates the persistence and activity of such clones.

[0111]

[0133] In another embodiment, the present invention relates to a method for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic population of TILs administered to a subject, wherein the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in a first population of PBMCs determined by RNA (e.g., mRNA) sequencing is compared with the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in a first population of PBMCs determined by DNA sequencing, wherein the frequency of such unique clones is greater when determined by RNA (e.g., mRNA) sequencing.

[0112]

[0134] The present invention's method for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a TIL therapeutic population administered to a subject involves determining the persistence and activity of T-cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones at various time points after administration of the TIL therapeutic population to the subject, for example, approximately 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and It can be applied to PBMCs isolated after 30 days, approximately 31 days, approximately 32 days, approximately 33 days, approximately 34 days, approximately 35 days, approximately 36 days, approximately 37 days, approximately 38 days, approximately 39 days, approximately 40 days, approximately 41 days, approximately 42 days, approximately 43 days, approximately 44 days, approximately 45 days, approximately 46 days, approximately 47 days, approximately 48 days, approximately 49 days, approximately 50 days, approximately 51 days, approximately 52 days, approximately 53 days, approximately 54 days, approximately 55 days, approximately 56 days, approximately 57 days, approximately 58 days, approximately 59 days, approximately 60 days, approximately 90 days, approximately 120 days, approximately 180 days, approximately 1 year, approximately 2 years, approximately 3 years, approximately 4 years, and / or approximately 5 years.

[0113] A system for identifying clinically effective populations for TIL

[0135] In another embodiment, the present invention relates to a system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), comprising: memory; one or more processors; and one or more modules stored in memory and configured for execution by one or more processors, the modules being (a) To identify T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones that constitute the clonal diversity of the TIL therapeutic population; (b) To identify T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones constituting the clonal diversity of a first population of peripheral blood mononuclear cells (PBMCs) isolated from the subjects at least 14 days after administration of the therapeutic population of step (a) to the subjects; (c) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (b), to determine the frequency of such unique TCR CDR3 clones in the therapeutic population of TIL and the first population of PBMCs, respectively; (d) To sort the unique TCR CDR3 coding nucleic acid sequence clones identified in step (b) from highest to lowest frequency for the therapeutic population of TIL and the first population of PBMCs, respectively; and (e) From the first population of PBMCs sorted in step (d), select 10 of the most frequently occurring unique TCR CDR3 coding nucleic acid sequence clones, such that TILs expressing such clones in the therapeutic population of TIL constitute a clinically effective subpopulation of TIL, thereby identifying a clinically effective population of TIL. This applies to systems that include the instruction.

[0114]

[0136] In another embodiment, the present invention relates to a system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), further comprising a module that includes instructions for carrying out: (i) identifying TCR CDR3 coding nucleic acid clones constituting the clonal diversity of a second population of PBMCs isolated from a subject prior to administration of a therapeutic population of TILs to the subject; and (ii) determining the frequency of each unique TCR CDR3 coding nucleic acid sequence clone identified in step (i).

[0115]

[0137] In another embodiment, the present invention relates to a system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), further comprising a module that includes instructions for comparing TCR CDR3 coding nucleic acid sequence clones constituting the CDR3 clonal diversity of a second population of PBMCs with TCR CDR3 coding nucleic acid sequence clones constituting the CDR3 clonal diversity of a first population of PBMCs.

[0116]

[0138] In another embodiment, the present invention relates to a system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), further comprising a module that includes instructions for determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones in at least one population based on DNA sequence data.

[0117]

[0139] In another embodiment, the present invention relates to a system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), further comprising a module that includes instructions for determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones in at least one population based on RNA sequence data.

[0118]

[0140] In another embodiment, the present invention relates to a system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), further comprising a module that includes instructions for determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones in at least one population based on both RNA sequence data and DNA sequence data.

[0119]

[0141] In another embodiment, the present invention relates to a system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), further comprising a module that includes instructions for comparing the frequency of unique TCR CDR3 coding nucleic acid clones in a first population of PBMCs determined by RNA sequencing with the frequency of unique TCR CDR3 coding nucleic acid clones in a first population of PBMCs determined by DNA sequencing, wherein the comparison targets a system that represents a clinically effective population of TILs. An exemplary module is shown in Figure 9 and generates a target-specific dataset by including all detected clones and clone frequencies in each sample. The results from this module can be processed by additional modules, also shown in Figure 9, to generate shared clone statistics, determine clone expression patterns, and determine persistent chronotypes. Further exemplary modules encode instructions for generating box plots, scatter plots, and heatmaps based on the output from other modules of the system.

[0120]

[0142] In another aspect, the present invention relates to a system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), further comprising at least one module which encodes instructions for manipulating data obtained from a TCR CDR3 coding nucleic acid clone, which is an mRNA clone, wherein such data is determined by RNA sequencing.

[0121] System for Sustainability

[0144] In another embodiment, the present invention relates to a system for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic population of TILs administered to a subject, comprising: memory; one or more processors; and one or more modules stored in memory and configured for execution by one or more processors, the modules being (a) To identify T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones that constitute the clonal diversity of the TIL therapeutic population; (b) To identify T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones constituting the clonal diversity of a first population of peripheral blood mononuclear cells (PBMCs) isolated from the subjects at least 14 days after administration of the therapeutic population of step (a) to the subjects; (c) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (b), to determine the frequency of such unique TCR CDR3 clones in the therapeutic population of TIL and the first population of PBMCs, respectively; and (d) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (b), the frequency of the TCR CDR3 coding nucleic acid sequence clone in the first population of PBMCs is compared with the frequency of the TCR CDR3 coding nucleic acid sequence clone in the TIL treatment population to determine the persistence and activity of the TCR CDR3 coding nucleic acid sequence clone in the treatment TIL population administered to the subjects. This applies to systems that include the instruction.

[0122]

[0145] In another embodiment, the present invention relates to a system for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic population of TILs, further modified to include a module comprising instructions for determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones in at least one population based on DNA sequence data.

[0123]

[0146] In another embodiment, the present invention relates to any system for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic population of TILs, which may be appropriately modified to further include a module comprising instructions for determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones in at least one population based on DNA sequence data.

[0124]

[0147] In another embodiment, the present invention relates to any system for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic population of TILs, which may be appropriately modified to further include a module comprising instructions for determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones in at least one population based on RNA sequence data.

[0125]

[0148] In another embodiment, the present invention relates to any system for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic population of TILs, which may be appropriately modified to further include a module comprising instructions for determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones in at least one population based on both RNA sequence data and DNA sequence data.

[0126]

[0149] In another embodiment, the present invention may be appropriately modified to further include a module containing instructions for comparing the frequency of unique TCR CDR3 coding nucleic acid clones in a first population of PBMCs determined by RNA sequencing with the frequency of unique TCR CDR3 coding nucleic acid clones in a first population of PBMCs determined by DNA sequencing, wherein the comparison applies to any system for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic TIL population, indicating the persistence and activity of TCR CDR3 coding nucleic acid sequence clones in the TIL therapeutic population. An exemplary module is shown in Figure 9 and generates a subject-specific dataset by including all detected clones and clone frequencies in each sample. The results from this module can be processed by additional modules, also shown in Figure 9, to generate shared clone statistics, determine clone expression patterns, and determine persistent chronotypes. Further exemplary modules code instructions for generating box plots, scatter plots, and heatmaps based on the output from other modules of the system.

[0127] Tumor type

[0150] In some embodiments, the method of the present invention utilizes early TILs expanded from tumor samples of cancer-affected subjects. Tumor samples can generally be obtained by surgical excision, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells, using methods known in the art. Generally, tumor samples may be from any solid tumor, e.g., primary tumor, invasive tumor, or metastatic tumor. Tumor samples may also be from humoral tumors, e.g., tumors obtained from hematological malignancies. Solid tumors may be from any type of cancer, e.g., but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma).

[0128]

[0151] In some embodiments, the subject has a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of melanoma (e.g., uveal melanoma), ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, basal cell carcinoma, cancer caused by human papillomavirus, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), brain cancer, glioblastoma (e.g., GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the solid tumor is melanoma. In some embodiments, the solid tumor is cervical cancer.

[0129]

[0152] In some embodiments, the subject suffers from a hematological malignancy or "humoral carcinoma." Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, and non-Hodgkin lymphoma. In some embodiments, the hematological malignancy is CLL. In some embodiments, the hematological malignancy is AML.

[0130]

[0153] It is understood that embodiments of the system of the present invention may only provide a portion of the aspects disclosed herein.

[0131]

[0154] Preferred embodiments of the present invention are shown and described herein, but such embodiments are provided only as examples and do not otherwise limit the scope of the invention. Various substitutes for the embodiments described may be used in carrying out the invention. [Examples]

[0132] Examples

[0155] The embodiments included herein are described below with reference to the following examples. These examples are provided for illustrative purposes only and should not be construed as limiting the disclosure contained herein to those examples, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0133] Example 1: uCDR3 clonal diversity in melanoma patients

[0156] In the C-144-01 trial for progressive metastatic melanoma, 25 patients with metastatic melanoma were selected to receive lifileucel (LN-144). The composition of circulating T cells was analyzed from the initial TIL product and 42 days after infusion to evaluate uCDR3 clonal diversity and TIL persistence.

[0134]

[0157] TIL products are polyclonal preparations of autologous T cells, and each T cell clone expresses a unique T cell receptor (TCR) that can be identified by complementarity-determining region 3 (CDR3). The CDR3 of TIL products and the corresponding post-infusion peripheral blood samples were subjected to RNA-seq using commercially available iRepertoire technology (Huntsville, AL). For a description of iRepertoire technology, see, for example, (i) Han, Jian, Method for Evaluating and Comparing Immunorepertoires, U.S. Patent No. 9012148, and (ii) Han, Jian, Method for Evaluating and Comparing Immunorepertoires, U.S. Patent No. 9012148, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0135]

[0158] Patient-borne microplastic tubes (PBMCs) were collected from each patient, and then personalized TIL treatments were administered. Patient PBMCs were collected again 42 days after the administration of the therapeutic TIL.

[0136]

[0159] For each sample, the T cell receptor (TCR) CDR3 diversity was determined in (1) personalized TIL treatment products; (2) pre-treatment PMBCs; and PBMCs recovered 42 days after TIL infusion. Nucleic acids were isolated and purified from each individual cell population. uCDR3 in each sample was identified using the isolated mRNA.

[0137]

[0160] After sequencing, a secondary analysis tool using Python version 3.6.3 was developed. All analyses and figures were generated using the Anaconda3 package and environmental management system. The steps for producing the data in Figures 2-7 are summarized below.

[0138]

[0161] Raw sequencing data was first analyzed preliminaryly to generate sample-specific summary CDR3 clone frequency data based on ray iRepertoire clonotype sequence data, and then the following were determined: (1) total read / CDR3 count was generated by sequencing; and (2) the unique CDR3 (uCDR3) clone count for each sample was calculated, followed by the Shannon diversity index. See Figure 2.

[0139]

[0162] Next, the system generated datasets for each subject: each of the first samples was normalized to a 10 million read baseline, and then a comprehensive list of clones and associated uCDR3 frequencies for each subject were generated for each sample. Next, the system analyzed shared clone statistics for each subject: the uCDR3 count for each sample was confirmed; and then the count of uCDR3 (shared uCDR3) found in the two samples was determined. These are the fitted TIL products and PBMC samples for each subject. Next, the sum of the frequencies of shared uCDR3 in each sample was calculated to determine the contribution of shared uCDR3 clones to the total uCDR3 clones in each sample.

[0140]

[0163] Next, the uCDR3 clone expansion pattern was determined. First, the 10 most frequent shared clones on day 42 were identified. Then, the percentile for each clone in the TIL product was determined, for example, clone 1 of 100 is 1%; clone 100 of 100 is 100%. See Figure 6. Finally, the system generated scatter plots of all percentiles for each subject.

[0141]

[0164] Next, the persistent uCDR3 chronotype was determined. First, a comprehensive list of persistent clones for each subject was combined with the persistent clone list for all subjects. Then, a clone heatmap was determined for subjects that highlighted persistent clones, see, for example, Figure 7.

[0142]

[0165] Finally, using various modules of the system, we produced comprehensive box plots to describe and characterize the total dataset, including each of the following plots: 1. TIL Shannon Entropy (Exponent) > Best Overall Effect, 2. TIL's unique CDR3 sequence (#) > Best overall result, 3. TIL's unique CDR3 arrangement (#)> works, 4. TIL Shannon Entropy (Exponent) > Success 5.TIL shared part > success, 6. D42 unique CDR3 arrangement (#)> effective, 7. D42 Shannon entropy (exponent) > Success 8.D42 shared part > success, 9. Shared TIL and D42uCDR3(#)> worked, 10. Shared uCDR3(%) in TIL > Success 11. Shared uCDR3(%) in D42 > Successful, 12. TIL's unique CDR3 arrangement (#) > reduction of diameter sum on day 42, 13. TIL Shannon Entropy (Exponent) > Reduction of the sum of diameters on day 42 14. TIL common part > reduction of diameter sum on day 42, 15. D42 Unique CDR3 arrangement (#) > Reduction of diameter sum on day 42 16. D42 Shannon entropy (exponent) > reduction of the sum of diameters on day 42, 17. D42 Shared portion > Reduction of diameter sum on day 42, 18. Reduction of shared TIL and D42uCDR3(#)> diameter sum on day 42, 19. Reduction of shared uCDR3(%) > diameter sum on day 42 in TIL, 20. Reduction of shared uCDR3(%) > diameter sum on day 42 in D42, 21. TIL's unique CDR3 arrangement (#)>DCR, 22. TIL Shannon Entropy (Exponent) > DCR, 23.TIL common part>DCR, 24. D42 unique CDR3 arrangement (#)>DCR, 25. D42 Shannon Entropy (Exponent) > DCR, 26.D42 common part>DCR, 27.Shared TIL and D42uCDR3(#)>DCR, 28. Shared uCDR3(%) > DCR in TIL, 29. Shared uCDR3(%) > DCR in D42

[0143]

[0166] Figure 2 shows the overall clonal diversity of non-responding patients (n=28) (left distribution in each plot) and responding patients (n=10) (right distribution in each plot). A detailed summary of the frequency of uCDR3 clones shared between TIL products and 42-day PBMCs is reported in Figure 3. The mean number of unique TCR CDR3 sequences (uCDR3) across TIL products was 17511 [3574~110797], and the Shannon diversity index varied from 2.7 to 10.8. Correlation analysis revealed no association between any parameter and clinical response, suggesting that tumor-responsive T cells are not affected by the level of diversity in bulk TILs (see Figures 4 and 5).

[0144]

[0167] 42 days after infusion, TIL clones were detectable in 100% of the patients' circulation, as shown in Figure 6. These shared uCDR3s were found at varying levels ranging from 28 to 6964 unique chronotypes, representing highly variable fractions of both TIL products and circulating T cells on day 42.

[0145]

[0168] The majority of shared uCDR3s were not detected in patients' peripheral blood at registration, indicating that they represent persistent intratumoral chronotypes after TIL administration. Furthermore, shared uCDR3 clones expressed at high or low frequencies in TIL products could persist for at least 6 weeks after infusion. Finally, Figures 6 and 7 show that over 97% of persistent clones were uniquely present in individual responding and non-responding patients, demonstrating a unique repertoire in each TIL preparation.

[0146] Example 2: Persistence of cryopreserved TIL products in patients with progressive melanoma.

[0170] Adoptive cell transplantation using tumor-infiltrating lymphocytes (TILs) is recognized as an effective treatment for metastatic melanoma and other solid tumors, inducing sustained and complete responses in patients with many prior treatment histories by presumably targeting tumor-specific somatic mutations (Rosenberg et al. CCR 2011). It has already been reported that patients with anti-PD-1 refractory progressive melanoma treated with rifireucel showed an overall response rate of 38% (SITC Nov2018). Here, we analyzed the composition of initial TIL products and circulating T cells (D42) 42 days after infusion to reveal potential links between clonal diversity, TIL in vivo persistence, and antitumor activity.

[0147]

[0171] Since TIL products are preparations of polyclonal autologous T cells, each T cell clone expresses a unique T cell receptor (TCR) that can be identified by complementarity-determining region 3 (CDR3). The CDR3 of TIL products and corresponding post-infusion peripheral blood samples were subjected to RNA-seq using iRepertoire technology (Huntsville, AL).

[0148]

[0172] The average number of unique TCR CDR3 sequences (uCDR3) across TIL products was 17511 [3574–110797], and the Shannon diversity index varied from 2.7 to 10.8. Correlation analysis revealed no association between any parameter and clinical response, suggesting that tumor-responsive T cells may exist in bulk TIL products with low and high diversity. At D42, TIL clones could be detected in the circulation of all treated patients. The number of shared uCDR3s ranged from 28 to 6964 unique chronotypes, representing highly variable fractions of both TIL products and D42 circulating T cells. A link between shared T cell clones and clinical response had already been hypothesized (Robbins et al. J Immunol 2004). In this study, similar proportions of shared uCDR3s were detected in both responders and non-responders. Importantly, the majority of shared uCDR3s were not detected in patients' peripheral blood at enrollment, indicating that they represented intratumoral chronotypes that persisted after TIL administration. Furthermore, shared uCDR3 clones expressed at high or low frequencies in TIL products could persist for at least 6 weeks after infusion. Finally, over 97% of persistent clones were uniquely present in individual responding and non-responding patients, demonstrating a unique repertoire in each TIL preparation.

[0149]

[0173] In summary, the data demonstrate that the TIL clone fraction persisted in all patients. The unique nature of the clonal profile associated with response highlights the difficulty in identifying a single TCR as a mediator of activity and supports the use of polyclonal products, e.g., bulk TILs, to treat solid tumors with associated unique, patient-specific, mutant, and neoantigen spectra.

[0150] Example 3: uCDR3 clonal diversity in cryopreserved TILs and persistence of TILs in progressive metastatic melanoma

[0174] Adoptive cell transplantation using tumor-infiltrating lymphocytes (TILs) is recognized as an effective treatment for metastatic melanoma and other solid tumors, inducing sustained and complete responses in patients with many prior treatment histories by presumably targeting tumor-specific somatic mutations (Rosenberg et al. CCR 2011). It has already been reported that patients with anti-PD-1 refractory progressive melanoma treated with rifireucel showed an overall response rate of 38% (SITC Nov2018). Here, we analyzed the composition of initial TIL products and circulating T cells (D42) 42 days after infusion to reveal potential links between clonal diversity, TIL in vivo persistence, and antitumor activity.

[0151]

[0175] Clinical trial C-144-01 is an ongoing phase 2 multicenter study investigating autologous TILs (also known as rifeureucel, LN-144). The patient population includes patients with unresectable metastatic melanoma that has progressed with checkpoint inhibitors and BRAF / MEK inhibitors. TILs were harvested, expanded, cryopreserved, and then prepared for infusion and infused into patients according to the 22-day process described in Figure 10. A total of 27 matched paired samples (i.e., one TIL product sample and one D42 PBMC sample from the same patient) were analyzed.

[0152]

[0176] Since TIL products are preparations of polyclonal autologous T cells, each T cell clone expresses a unique T cell receptor (TCR) that can be identified by complementarity-determining region 3 (CDR3). Total RNA was extracted using the Qiagen RNeasy Mini Kit Protocol. CDR3 from TIL products and corresponding post-infusion peripheral blood samples was subjected to RNA amplification and sequencing using iRepertoire technology (Huntsville, AL). Target CDR3 clones were identified using a custom Python script, and statistical analysis was performed. After sequencing, unique CDR3 sequence counts were calculated and plotted in Figure 11A. Correlation analysis suggested no association between the number or diversity score of TCR chronotypes and clinical response. Non-responders (n=17) and responders (n=10) showed similar results (p=0.2447). Shannon entropy was also calculated for the same samples and plotted in Figure 11B. Here again, non-responders (n=17) and responders (n=10) showed similar results (p=0.2499). The median values ​​for both variables are shown by horizontal lines in each box plot. The groups are based on response criteria in solid tumors, with the responder group including subjects with partial or complete response and the non-responder group including subjects with stable or progressive disease. The mean number of unique TCR CDR3 sequences (uCDR3) across TIL products was 17511 [3574~110797], and the Shannon diversity index varied from 2.7 to 10.8. The lack of correlation between the number of chronotypes and clinical response suggests that tumor-reactive T cells may be present in bulk TIL products with low and high diversity. This supports the idea that bulk TIL products can recover relevant TILs without prior knowledge of tumor antigens.

[0153]

[0177] The number of shared CDR3s was determined by measuring the number of detected CDR3 clones in circulation (D42 samples) that were also present in the corresponding TIL products. Shared CDR3s were detected in all D42 samples analyzed at varying levels of 28 to approximately 6900 clones. Figure 12A illustrates the number of clones in the initial TIL product (black bars) and the D42 samples (gray bars). Clonal frequencies were calculated for the TIL product, D42 samples, and pre-infusion PBMCs to assess whether TIL clones were already present in the patient's blood. For this particular test, n=15. The results are shown in Figure 12B as a bar graph of the sum of shared clone frequencies expressed as a percentage. Of the 29,745 shared clones identified, 69% (or 20,480) were undetectable before infusion. This suggests that (i) in vivo persistent TIL clones are either not present in the blood prior to TIL infusion, or are present at a very low frequency, and (ii) TIL product expansion is tumor antigen specific.

[0154]

[0178] The top 10 persistent clones were evaluated and ranked in TIL products and D42 samples. Each point in Figure 13 represents the rank as a percentage within the TIL product for each of the top-ranking clones for each patient. The most frequent clone in the TIL product corresponds to the minimum value; the least frequent clone corresponds to the maximum value (closer to 100). Persistent clones were found at both high and low frequencies in the TIL product, and the uCDR3 clone could persist for at least 6 weeks after infusion, regardless of its high or low frequency in the TIL product. This data suggests that the abundance of clones in the TIL product does not correlate with their abundance at the circulating D42 level in the blood. This indicates the periodic expansion of TILs in vivo as clones encounter their cognitive antigens.

[0155]

[0179] The correlation between persistent T cell clones and clinical response was also analyzed. The proportion of persistent clones was determined by dividing the number of clones detected in both the TIL product and the D42 sample by the number of uCDR3 clones in the TIL product. A measure of overlap or shared uCDR3 between the TIL product and in vivo circulating T cells on day 42 (expressed as a percentage, p=0.0484) is shown as a box plot in Figure 14 for non-responders (n=17) and responders (n=10). The median of both variables is shown by a horizontal line in each box plot. This data suggests that clinical response may be associated with in vivo TIL persistence.

[0156]

[0180] A total of 47,508 persistent clones were identified from 27 subjects. CDR3 alignment revealed that 45,944 sequences (96.7%) were found in only one subject (see Figure 15A). 45 sequences were found in more than four subjects; 17 of these (37.8%) corresponded to CDR3s already identified to recognize non-tumor-associated epitopes (e.g., CMV, EBV, influenza, etc.) (see https: / / vdjdb.cdr3.net). Figure 15B illustrates this data in tabular form. The data suggest that there is no correlation between clonal commonality and clinical response, and that the T cell repertoire, e.g., potentially tumor-specific clones, are unique to each TIL preparation.

[0157]

[0181] In summary, the data support the use of polyclonal products, such as bulk TIL products, for treating solid tumors with relevant, unique, patient-specific, mutant, and neoantigen spectra. The data demonstrate that 100% of TIL products produced by the process described in Figure 10 demonstrate levels of in vivo persistence six weeks after infusion. Although TIL products are highly polyclonal, neither the number of unique clones nor the diversity index is associated with clinical response. The in vivo fate of individual T cell clones is not thought to depend on their frequency in the infusion product, but rather reflects their specific antigenic reactivity. Furthermore, TIL products are highly specific to each patient and contain a unique TCR repertoire.

[0158] Example 4: In vivo sustained efficacy of tumor-infiltrating lymphocyte (TIL) product LN-145 in cervical cancer patients.

[0182] Clinical trial C-145-04 (NCT03108495) is an ongoing phase 2 multicenter study investigating autologous TILs (also known as LN-145). Patients with metastatic, recurrent, or persistent cervical cancer were treated with an ex vivo expanded autologous TIL product (LN-145). The overall response rate was 44%, and the disease control rate was 85%.

[0159]

[0183] We analyzed initial TILs in pre-infusion LN-145 products and T cells circulating in the blood 42 days after infusion (D42) to clarify the relationship between clonal diversity, TIL in vivo persistence, and antitumor activity. Each T cell clone in the TIL product expresses a unique T cell receptor identifiable by complementarity-determining region 3 (CDR3). Unique CDR3 sequences (uCDR3) and Shannon entropy for these T cell clones were identified using the methods discussed in the above examples. Peripheral blood from initial LN-145 TILs from study C-145-04 and corresponding D42 infusions from the same patient were subjected to CDR3 RNA sequencing (iRepertoire, Huntsville, AL). Pre-infusion LN-145 uCDR3 counts and Shannon diversity indices showed high variability (1,167–61,167 and 4.8–11.4, respectively) in both the overall response rate and disease control rate cohorts, suggesting that both low and high diversity LN-145 could contain tumor-responsive T cells. In D42 peripheral blood samples, a mean of 2079 LN-145-derived clones were present in all patients, representing 12% and 20% of the LN-145 and D42 blood uCDR3, respectively. However, these shared uCDR3s represented a significant portion of the total CDR3 repertoire in both LN-145 (62%) and D42 samples (52%). Overlap between pre-infusion LN-145 and D42 blood did not correlate with clinical response. Most clones common to LN-145 and D42 were not detected in peripheral blood at registration, indicating their presumed LN-145 origin and post-infusion persistence. The frequency of each persistent clone in the initial TIL product did not predict their prevalence in D42 blood. Finally, over 98% (32,329 / 32,757) of the persistent clones were specific to individual responding and non-responding patients, demonstrating a unique repertoire in each TIL preparation.

[0160]

[0184] In summary, TIL clone fractionation persisted in all patients in this highly successful population. The unique nature of each TIL product and its associated clonal profile highlights the difficulty in identifying a single TCR as a common mediator of activity and supports the use of polyclonal products, e.g., bulk TILs, to treat solid tumors with associated private, patient-specific, mutant, and neoantigen spectra.

[0161] Example 5: Use of TCR repertoire analysis to determine TIL production process equivalence

[0185] Since TIL products are preparations of polyclonal autologous T cells, each T cell clone expresses a unique T cell receptor (TCR) that can be identified by complementarity-determining region 3 (CDR3). The therapeutic efficacy of a TIL population may correlate with the CDR3 clonal diversity of TILs in that population. However, minor variability in the manufacturing process between institutions may affect the CDR3 clonal diversity of TIL populations and, therefore, their therapeutic efficacy. Thus, a method for determining the equivalence of TIL populations produced at different institutions is desired. In one embodiment, the present disclosure provides a method for determining TIL production process equivalence using TCR repertoire analysis, comprising: determining the CDR3 clonal diversity of TIL in a first sample, e.g., a first set of unique CDR3 sequences expressed by TIL in the first sample; determining the CDR3 clonal diversity of TIL in a second sample, e.g., a second set of unique CDR3 sequences expressed by TIL in the second sample; determining the number of unique CDR3 sequences occurring in both the first and second sets; (i) determining the ratio of the number of unique CDR3 sequences occurring in both the first and second sets to the number of unique CDR3 sequences in the first set, and / or (ii) determining the ratio of the number of unique CDR3 sequences occurring in both the first and second sets to the number of unique CDR3 sequences in the second set; and determining the equivalence of TIL in the first sample and TIL in the second sample based on the ratio.

[0162]

[0186] To test the equivalence of the TIL production process, T cells underwent a pre-rapid expansion process followed by rapid expansion at three independent facilities, as previously described (Figure 16). Total RNA was extracted from each sample using the Qiagen RNeasy Mini Kit Protocol. The CDR3 of the TIL product was subjected to RNA amplification and sequencing using iRepertoire technology (Huntsville, AL). A custom Python script was used to identify the target CDR3 clone and perform statistical analysis. After sequencing, the unique CDR3 sequence count was calculated and is shown in Figure 17. To compare the CDR3 clonal diversity of TILs in samples produced at different facilities (e.g., Sample 1 and Sample 2), the ratio of the number of unique CDR3 sequences shared by both samples to the number of unique CDR3 sequences in Sample 1 was determined. Similarly, the ratio of the number of unique CDR3 sequences shared by both samples to the number of unique CDR3 sequences in Sample 2 was also determined. The obtained values ​​are a measure of the similarity of CDR3 clonal diversity between two samples, with values ​​of at least 10% indicating similarity of CDR3 clonal diversity between samples in one embodiment. Similar results were obtained when analyzing iRepertoire data from peripheral blood lymphocyte (PBL) samples, as shown in Figure 18. Specifically, the ratio of the number of unique CDR3 sequences shared by samples produced at two different institutions to the number of unique CDR3 sequences in either sample was determined to be greater than approximately 15%, indicating similarity of CDR3 clonal diversity between samples. In contrast, as shown in Figures 20, 22, and 23, values ​​of less than approximately 2% indicated that the samples were unrelated (e.g., the samples had low equivalence or similarity of CDR3 clonal diversity).

Claims

1. A method for identifying a clinically effective population of tumor-infiltrating lymphocytes (TILs) in a TIL treatment population administered to a subject, (i) Identify TCR CDR3 coding nucleic acid sequence clones that constitute T cell receptor (TCR) complementarity-determining region 3 (CDR3) clonal diversity in the TIL treatment population; (ii) Identifying TCR-CDR3 coding nucleic acid sequence clones constituting the TCR-CDR3 clonal diversity of a first population of peripheral blood mononuclear cells (PBMCs) isolated from a subject at least 14 days after administration of the therapeutic population of step (i) to the subject; (iii) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (iii), determine the frequency of such unique TCR CDR3 clones in the therapeutic population of TIL and the first population of PBMC, respectively; (iv) Sort the unique TCR CDR3 coding nucleic acid sequence clones identified in step (ii) from highest frequency to lowest frequency for each of the therapeutic population of TIL and the first population of PBMC; and (v) Select from the first population of PBMCs sorted in step (iv) 10 most frequent unique TCR CDR3 coding nucleic acid sequence clones such that TILs expressing such clones in the therapeutic population of TIL constitute a clinically effective population of TIL, thereby identifying the clinically effective population of TIL. Includes, A method by which a clinically effective population provides a clinically detectable therapeutic and / or preventive effect to the subject by administering an early-stage TIL population.

2. The method according to claim 1, further comprising: (a) identifying TCR CDR3 coding nucleic acid sequence clones constituting the TCR CDR3 clonal diversity of a second population of PBMCs isolated from the subject prior to administration of the therapeutic population of TIL to the subject; and (b) determining the frequency of each unique TCR CDR3 coding nucleic acid sequence clone identified in step (a).

3. The method according to claim 2, wherein the TCR CDR3 coding nucleic acid sequence clones constituting the clonal diversity of the second population of PBMCs are different from the TCR CDR3 coding nucleic acid sequence clones constituting the clonal diversity of the first population of PBMCs isolated from the subjects after administration of the therapeutic population of TIL.

4. The method according to any one of claims 1 to 3, wherein the frequency of a unique TCR CDR3 coding nucleic acid sequence clone identified in at least one population is determined by DNA sequencing and / or RNA sequencing.

5. The method according to any one of claims 1 to 3, wherein the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in the therapeutic population of TIL and the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in the first population of PBMCs is determined by both DNA and RNA sequencing, the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in the first population of PBMCs as determined by RNA sequencing is compared with the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in the first population of PBMCs as determined by DNA sequencing, and the frequency of such unique clones determined by RNA sequencing indicates a clinically effective population of TIL compared with the frequency of such unique clones determined by DNA sequencing.

6. The method according to claim 5, wherein the frequency of such unique clones is large when determined by RNA sequencing and / or DNA sequencing.

7. The method according to claim 6, wherein the frequency of such unique clones determined by RNA sequencing correlates with a population of TILs having improved therapeutic efficacy, and / or the frequency of such unique clones determined by DNA sequencing does not correlate with a population of TILs having improved therapeutic efficacy.

8. The method according to any one of claims 1 to 7, wherein the TCR CDR3 coding nucleic acid sequence clone is an mRNA clone identified by RNA sequencing.

9. The method according to any one of claims 1 to 8, wherein the mRNA can be detected during a period selected from the groups consisting of 20, 25, 30, 35, 40, 42, 45, 50, 55, 60, 90, 120, 180 days, 1 year, and 2 years after administration to the TIL treatment population.

10. An ex vivo method for improving T cell repertoire, (i) Identifying a clinically effective population of tumor-infiltrating lymphocytes by the method described in any one of claims 1 to 9; and (ii) Select and expand the population identified in step (i) to produce a second clinically effective therapeutic population for TIL. A method that includes this.

11. A system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), Memory; One or more processors; and It includes one or more modules stored in memory and configured for execution by one or more processors, and the modules are (a) To identify T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones that constitute the clonal diversity of the TIL treatment population; (b) To identify T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones that constitute the clonal diversity of a first population of peripheral blood mononuclear cells (PBMCs) isolated from a subject at least 14 days after administration of the therapeutic population of step (a) to the subject; (c) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (b), to determine the frequency of such unique TCR CDR3 clones in the therapeutic population of TIL and the first population of PBMC, respectively; (d) To sort the unique TCR CDR3 coding nucleic acid sequence clones identified in step (b) from highest frequency to lowest frequency for each of the therapeutic population of TIL and the first population of PBMC; and (e) Select from the first population of PBMCs sorted in step (d) 10 most frequent unique TCR CDR3 coding nucleic acid sequence clones such that TILs expressing such clones in the therapeutic population of TILs constitute a clinically effective subpopulation of TILs, thereby identifying the clinically effective population of TILs. Includes instructions, A system in which a clinically effective population, by administering an early-stage TIL population, brings about a clinically detectable therapeutic and / or preventive effect in the subject.

12. The module further includes instructions for carrying out the steps of (x) identifying TCR CDR3 coding nucleic acid clones that constitute the clonal diversity of a second population of PBMCs isolated from the subject prior to administration of the TIL to the subject; and (y) determining the frequency of each unique TCR CDR3 coding nucleic acid sequence clone identified in step (x); or The system according to claim 11, further comprising a module that includes instructions for comparing the TCR CDR3 coding nucleic acid sequence clones constituting the CDR3 clonal diversity of a second population of PBMCs with the TCR CDR3 coding nucleic acid sequence clones constituting the CDR3 clonal diversity of a first population of PBMCs.

13. The system according to claim 11 or 12, further comprising a module that includes instructions for determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones in at least one population based on DNA sequence data and / or RNA sequence data.

14. The above instructions are for determining the frequency of unique TCR CDR3 coding nucleic acid sequence clones in at least one population based on both RNA sequence data and DNA sequence data, The system according to claim 13, further comprising a module that includes instructions for comparing the frequency of unique TCR CDR3 coding nucleic acid clones in the first population of PBMCs determined by RNA sequencing with the frequency of unique TCR CDR3 coding nucleic acid clones in the first population of PBMCs determined by DNA sequencing, wherein the comparison indicates the clinically effective population of TILs.

15. The system according to any one of claims 11 to 14, wherein the TCR CDR3 coding nucleic acid clone is an mRNA clone identified by RNA sequencing.

16. A method for determining the persistence and activity of T cell receptor (TCR) complementarity-determining region 3 (CDR3) coding nucleic acid sequence clones in tumor-infiltrating lymphocytes (TILs) in a therapeutic population of TILs administered to a subject, (a) Identify the TCR-CDR3 coding nucleic acid clones that constitute the TCR-CDR3 clonal diversity in the TIL treatment population; (b) Identifying TCR CDR3 coding nucleic acid sequence clones that constitute the TCR CDR3 clonal diversity of a first population of peripheral blood mononuclear cells (PBMCs) isolated from a subject at least 14 days after administration of the therapeutic population of step (a) to the subject; (c) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (b), determine the frequency of such unique TCR CDR3 coding nucleic acid sequence clones in the therapeutic population of TIL and the first population of PBMC, respectively; and (d) For each unique TCR CDR3 coding nucleic acid sequence clone identified in step (b), the frequency of the TCR CDR3 coding nucleic acid sequence clone in the first population of PBMCs is compared with the frequency of the TCR CDR3 coding nucleic acid sequence clone in the therapeutic TIL population to determine the persistence and activity of the TCR CDR3 coding nucleic acid sequence clone in the therapeutic TIL population administered to the subjects. Includes, A method in which the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in the therapeutic population of TIL and the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in the first population of PBMC are determined by RNA sequencing.

17. The method according to claim 16, further comprising determining by DNA sequencing the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in the therapeutic population of TIL and the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in the first population of PBMC.

18. The method according to claim 17, wherein the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in the first population of PBMCs determined by RNA sequencing is compared with the frequency of unique TCR CDR3 coding nucleic acid sequence clones identified in the first population of PBMCs determined by DNA sequencing, and the frequency of such unique clones determined by RNA sequencing compared with the frequency of such unique clones determined by DNA sequencing indicates the persistence and activity of such clones.

19. The method according to claim 18, wherein the frequency of such unique clones is large when determined by RNA sequencing.

20. A method for identifying a clinically effective population of tumor-infiltrating lymphocytes (TILs) in subjects treated with a TIL therapy population, (i) Determining the CDR3 clonal diversity of the TIL treatment population; (ii) Determining the CDR3 clonal diversity of peripheral blood mononuclear cells (PBMCs) isolated from the subjects at least 14 days after administration of the therapeutic population of step (i) to the subjects; (iii) Identify the CDR3 clones identified in both steps (i) and (ii); (iv) Sort the CDR3 clones identified in step (iii) from highest frequency to lowest frequency with respect to the therapeutic population of TIL and the PBMC, respectively; and (iv) Select the 10 most frequent CDR3 clones from step (iv) identified in the PBMC, thereby identifying a clinically effective population of TILs. Includes, A method by which a clinically effective population provides a clinically detectable therapeutic and / or preventive effect to the subject by administering an early-stage TIL population.

21. A system for identifying clinically effective populations of tumor-infiltrating lymphocytes (TILs), Memory; One or more processors; and It includes one or more modules stored in memory and configured for execution by one or more processors, and the modules are (a) To determine the CDR3 clonal diversity of the TIL treatment population; (b) To determine the CDR3 clonal diversity of peripheral blood mononuclear cells (PBMCs) isolated from subjects at least 14 days after administration of the therapeutic population of step (a) to the subjects; (c) To identify the CDR3 clone identified in both steps (a) and (b); (d) To sort the CDR3 clones identified in step (c) from highest frequency to lowest frequency with respect to the therapeutic population of TIL and the PBMC, respectively; and (e) Select the 10 most frequent CDR3 clones from step (d) identified in the PBMC to identify a clinically effective population of TILs. Includes instructions, A system in which a clinically effective population, by administering an early-stage TIL population, brings about a clinically detectable therapeutic and / or preventive effect in the subject.

22. The subject is a solid tumor carcinoma, and the method according to any one of claims 1 to 10 or the system according to any one of claims 11 to 21.

23. The method according to claim 22, wherein the solid tumor cancer is selected from the group consisting of melanoma (e.g., uveal melanoma), ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, basal cell carcinoma, cancer caused by human papillomavirus, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), brain cancer glioblastoma (e.g., GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma.

24. The method according to claim 22, wherein the cancer is melanoma or cervical cancer.

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