Compositions and methods for T cell receptor gene assembly

Abstract

Provided herein are compositions and methods for assembling nucleic acid sequences encoding T cell receptors.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 829,813 filed on April 5, 2019, U.S. Provisional Patent Application No. 62 / 838,465 filed on April 25, 2019, U.S. Provisional Patent Application No. 62 / 898,053 filed on September 10, 2019, and U.S. Provisional Patent Application No. 62 / 972,231 filed on February 10, 2020, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on May 15, 2020, is named 53563-705_601_SL.txt, and is 69,187 bytes in size. Background Art

[0005] T cell receptor (TCR) can be responsible for identifying antigen-major histocompatibility complex, thereby leading to the initiation of inflammatory response. There are many T cell subsets, including cytotoxic T cells and helper T cells. Cytotoxic T cells (also known as CD8+T cells) kill abnormal cells, such as virus-infected cells or tumor cells. Helper T cells (also known as CD4+T cells) contribute to the activation and maturation of other immune cells. Both cytotoxic T cells and helper T cells perform their functions after recognizing specific target antigens, thereby triggering their respective reactions. The antigen specificity of T cells can be defined by the TCR expressed on the surface of T cells. The T cell receptor is a heterodimeric protein composed of two polypeptide chains, the most common of which is α chain and β chain, but a few T cells can express γ and δ chains. The specific amino acid sequence of TCR and the resulting three-dimensional structure define TCR antigen specificity and affinity. The amino acid and encoding DNA sequence of the TCR chain of any single T cell are almost always unique or very low in abundance in the entire TCR library of the organism, because there are a large number of possible TCR sequences. This enormous sequence diversity can be achieved by multiple cellular mechanisms during T cell development and may be a key aspect of the immune system's ability to respond to a wide range of potential antigens.

[0006] Analysis of the TCR repertoire can help to better understand the characteristics of the immune system and the etiology and progression of diseases, especially those with unknown antigenic triggers. The extreme diversity of the TCR repertoire and the dichotomous nature of TCRs can represent major analytical challenges. High-throughput sequencing can allow greater sequencing depth and significantly more accurate quantification of TCR clonotype abundance, although at a higher cost than spectral profiling. Summary of the invention

[0007] Provided herein are compositions and methods for assembling nucleic acid sequences encoding naturally paired T cell receptors (TCRs) (or homologous TCR pairs). For example, a TCR may include a TCR alpha chain and a TCR beta chain, or a TCR may include a TCR gamma chain and a TCR delta chain. Sequences encoding naturally paired TCRs can be identified using various methods, including but not limited to using single cell barcodes and sequencing techniques. After obtaining sequences encoding naturally paired TCRs, the compositions and methods described herein can be used to construct or assemble one or more nucleic acid sequences to express naturally paired TCRs in any given host cell in a fast, high throughput and cost-effective manner. The one or more nucleic acid sequences may comprise greater than or equal to about 1, 5, 10, 20, 50, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 15,000, 20,000, 100,000, 1,000,000, 10,000,000 or more different sequences encoding different TCRs.

[0008] In one aspect, the present disclosure provides a method for producing a nucleic acid molecule encoding a T cell receptor (TCR) chain or a portion thereof, comprising: (a) providing at least one nucleic acid molecule comprising a sequence encoding CDR3 of the TCR chain; (b) providing a plurality of nucleic acid molecules, each of the plurality of nucleic acid molecules comprising a sequence derived from a TCR V gene, wherein the plurality of nucleic acid molecules comprise at least two different sequences derived from at least two different TCR V genes; and (c) contacting at least one nucleic acid molecule of (a) with a plurality of nucleic acid molecules of (b) in the same compartment, wherein the at least one nucleic acid molecule of (a) is capable of connecting to a nucleic acid molecule in the plurality of nucleic acid molecules to produce a third nucleic acid molecule comprising a sequence encoding CDR3 and a sequence derived from one of at least two different TCR V genes, thereby producing a nucleic acid molecule encoding a TCR chain or a portion thereof. In some embodiments, at least one nucleic acid molecule comprises at least about 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 15,000, 20,000, 100,000, 1,000,000, 10,000,000 or more different sequences. In some embodiments, the plurality of nucleic acid molecules (each of the plurality of nucleic acid molecules comprises a nucleic acid molecule derived from a TCR 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or more V genes derived from different TCRs Different sequences of V genes.

[0009] In some embodiments, at least one nucleic acid molecule comprises a first plurality of nucleic acid molecules, wherein each nucleic acid molecule in the first plurality of nucleic acid molecules comprises a sequence of a CDR3 encoding a TCR chain. In some embodiments, at least one nucleic acid molecule of (a) can be specifically connected to a nucleic acid molecule in the plurality of nucleic acid molecules, and the nucleic acid molecule in the plurality of nucleic acid molecules comprises a sequence of any single given TCR V gene derived from at least two different TCR V genes. In some embodiments, at least one nucleic acid molecule also comprises the J region of a TCR chain. In some embodiments, each nucleic acid molecule in the first plurality of nucleic acid molecules also comprises the J region of a TCR chain. In some embodiments, the at least two TCR V genes are human TCR V genes or mouse TCR V genes. In some embodiments, the at least two TCR V genes are selected from human TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV7, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8-6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV13-2, TRAV14 , TRAV16, TRAV17, TRAV18, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23, TRAV24, TRAV25, TRAV26-1, TRAV26-2, TRAV27, TRAV29, TRAV30, TRAV34, TRAV35, TRAV36, TRAV38-1, TRAV38-2, TRAV39, TRAV40 and TRAV41.In some embodiments, the at least two TCR V genes are selected from human TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV4-3, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV5-8, TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV6-9, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-8, The group consisting of TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1 and TRBV30. In some embodiments, each of the multiple sequences derived from at least two different TCR V genes comprises a sequence encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and / or FR3. In some embodiments, the TCR chain is a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. In some embodiments, at least one nucleic acid molecule further comprises an additional sequence of an additional CDR3 encoding an additional TCR chain. In some embodiments, at least one nucleic acid molecule comprises an additional J region of an additional TCR chain. In some embodiments, the sequence encoding CDR3 and the additional sequence encoding additional CDR3 are separated by at most 100 nucleotides. In some embodiments, the TCR chain and the additional TCR chain are homologous pairs of TCR chains. In some embodiments, at least one nucleic acid molecule comprises a connection sequence, and the connection sequence can connect at least one nucleic acid molecule to a nucleic acid molecule in a plurality of nucleic acid molecules to produce a third nucleic acid molecule. In some embodiments, the nucleic acid molecules in at least one nucleic acid molecule and a plurality of nucleic acid molecules encode functional TCR chains or parts thereof. In some embodiments, the nucleic acid molecules in a plurality of nucleic acid molecules comprise an anti-connection sequence, and the anti-connection sequence is complementary to the connection sequence of at least one nucleic acid molecule of (a). In some embodiments, the method further comprises connecting the nucleic acid molecules in at least one nucleic acid molecule of (a) and a plurality of nucleic acid molecules of (b). In some embodiments, the connection comprises hybridizing the at least one nucleic acid molecule of (a) with a nucleic acid molecule in the plurality of nucleic acid molecules of (b). In some embodiments, the hybridization comprises hybridizing the connection sequence of the at least one nucleic acid molecule of (a) with an anti-connection sequence of a nucleic acid molecule in the plurality of nucleic acid molecules of (b).In some embodiments, the method further comprises (i) using at least one nucleic acid molecule of (a) as a template to extend the free 3' end of the nucleic acid molecule in the plurality of nucleic acid molecules, and / or (ii) using the nucleic acid molecule in the plurality of nucleic acid molecules as a template to extend the free 3' end of at least one nucleic acid molecule of (a) to produce a third nucleic acid molecule. In some embodiments, the method further comprises connecting at least one nucleic acid molecule of (a) to the nucleic acid molecule in the plurality of nucleic acid molecules of (b). In some embodiments, the method further comprises contacting the third nucleic acid molecule with a restriction enzyme to produce a sticky end. In some embodiments, the method further comprises contacting the third nucleic acid molecule with an additional nucleic acid molecule. In some embodiments, the additional nucleic acid molecule encodes the constant region or part thereof of the TCR chain. In some embodiments, the method further comprises connecting the third nucleic acid molecule with an additional nucleic acid molecule. In some embodiments, a plurality of nucleic acid molecules (each encoding a different TCR chain or part thereof) are produced in the same compartment. In some embodiments, at least five different nucleic acid molecules in the plurality of nucleic acid molecules are produced in the same compartment. In some embodiments, at least ten different nucleic acid molecules in the plurality of nucleic acid molecules are produced in the same compartment. In some embodiments, at least 20, 50, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 15,000, 20,000, 100,000, 1,000,000, 10,000,000 or more different nucleic acid molecules in a plurality of nucleic acid molecules are produced in the same compartment. In some embodiments, the same compartment is a well, a tube or a droplet. In some embodiments, at least one nucleic acid molecule comprises a unique barcode. In some embodiments, the unique barcode is a primer binding site. In some embodiments, the connection sequence comprises a unique barcode. In some embodiments, the unique barcode is a primer binding site.

[0010] On the other hand, the present disclosure provides a composition comprising (a) a plurality of nucleic acid molecules, wherein each of the plurality of nucleic acid molecules comprises a sequence derived from a T cell receptor (TCR) V gene and does not comprise a CDR3 sequence, wherein a first nucleic acid molecule in the plurality comprises a first anti-linking sequence and a second nucleic acid molecule in the plurality comprises a second anti-linking sequence, wherein the first anti-linking sequence is different from the second anti-linking sequence, and wherein the sequences derived from the TCR V gene of the first nucleic acid molecule and the second nucleic acid molecule are derived from different TCR V genes; and (b) at least one nucleic acid molecule comprising a sequence encoding the CDR3 of the TCR chain, wherein the at least one nucleic acid molecule further comprises a first linking sequence complementary to the first anti-linking sequence.

[0011] In some embodiments, composition is liquid composition.In some embodiments, (a) multiple nucleic acid molecules and (b) at least one nucleic acid molecule are in the same compartment.In some embodiments, the sequence derived from TCR V gene comprises at least ten nucleotides of TCR V gene.In some embodiments, TCR V gene is TRAV gene, TRBV gene, TRGV gene or TRDV gene.In some embodiments, the sequence derived from TCR V gene comprises the sequence of encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and / or FR3.In some embodiments, at least one nucleic acid molecule also comprises the J district of TCR chain.In some embodiments, at least one nucleic acid molecule also comprises the additional sequence of the additional CDR3 of encoding additional TCR chain.In some embodiments, at least one nucleic acid molecule also comprises the additional J district of additional TCR chain.In some embodiments, the sequence encoding CDR3 and the additional sequence encoding CDR3 are separated by at most 100 nucleotides.In some embodiments, TCR chain and additional TCR chain are the homology pairs of TCR chain. In some embodiments, at least one nucleic acid molecule of (b) comprises a first plurality of nucleic acid molecules, and each nucleic acid molecule in the first plurality of nucleic acid molecules comprises a sequence encoding the CDR3 of a TCR chain. In some embodiments, each nucleic acid molecule in the first plurality of nucleic acid molecules encodes different CDR3s of different TCR chains. In some embodiments, each nucleic acid molecule in the first plurality of nucleic acid molecules comprises different connection sequences, and the different connection sequences can be specifically connected to a nucleic acid molecule comprising a sequence derived from any single given TCR V gene in a plurality of nucleic acid molecules. In some embodiments, the first anti-connection sequence or the second anti-connection sequence comprises a TCR V gene sequence. In some embodiments, the TCR V gene sequence comprises at least three nucleotides of a TCR V gene adjacent to a sequence encoding CDR3 in a rearranged gene. In some embodiments, the first anti-connection sequence or the second anti-connection sequence comprises a predetermined sequence. In some embodiments, the first connection sequence hybridizes with the first anti-connection sequence. In some embodiments, at least one nucleic acid molecule of (b) comprises a unique barcode. In some embodiments, a unique barcode is a primer binding site. In some embodiments, the first connection sequence of at least one nucleic acid molecule comprises a unique barcode. In some embodiments, the unique barcode is a primer binding site.

[0012] On the other hand, the present disclosure provides a method for producing multiple nucleic acid molecules, comprising: providing a first plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules comprise sequences encoding a first CDR3 of a first T cell receptor (TCR) chain and a second CDR3 of a second TCR chain, wherein the first CDR3 and the second CDR3 are from a homologous pair of TCR chains; providing a second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the second plurality of nucleic acid molecules comprise sequences derived from TCR V genes, wherein the nucleic acid molecules do not comprise sequences encoding constant domains; and contacting the first plurality of nucleic acid molecules with the second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules are connected to the nucleic acid molecules in the second plurality of nucleic acid molecules to form a nucleic acid molecule comprising a sequence encoding the first CDR3 and the second CDR3 and a sequence derived from the TCR V gene, wherein the sequences encoding the first CDR3 and the second CDR3 and the TCR V gene are derived from a homologous pair of TCR chains.

[0013] In some embodiments, every kind of nucleic acid molecule in the first multiple nucleic acid molecule includes the sequence of the different first CDR3 and / or the different CDR3 of the second TCR chain of encoding the first TCR chain.In some embodiments, the first multiple nucleic acid molecule includes at least about 2,5,10,20,50,100,200,300,400,500,1,000,1,500,2,000,2,500,3,000,3,500,4,000,5,000,6,000,7,000,8,000,9,000,10,000,12,000,15,000,20,000,100,000,1,000,000,10,000,000 or more different sequences.In some embodiments, every kind of nucleic acid molecule in the second multiple nucleic acid molecule includes the sequence derived from different TCR V genes. In some embodiments, the second plurality of nucleic acid molecules comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or more different TCRs. V gene. In some embodiments, the first multiple nucleic acid molecules and the second multiple nucleic acid molecules are contacted in the same compartment. In some embodiments, the nucleic acid molecules in the first multiple nucleic acid molecules also include a connecting sequence, wherein the connecting sequence connects the nucleic acid molecules in the first multiple nucleic acid molecules to the nucleic acid molecules in the second multiple nucleic acid molecules. In some embodiments, the nucleic acid molecules in the second multiple nucleic acid molecules also include an anti-connection sequence, which is complementary to the connecting sequence. In some embodiments, the connecting sequence hybridizes with the anti-connection sequence to connect the nucleic acid molecules in the first multiple nucleic acid molecules to the nucleic acid molecules in the second multiple nucleic acid molecules. In some embodiments, the connecting sequence is codon-diverse, so that the connecting sequence of the nucleic acid molecules in the first multiple nucleic acid molecules is different from the other connecting sequences of other nucleic acid molecules in the first multiple nucleic acid molecules. In some embodiments, the nucleic acid molecules in the first multiple nucleic acid molecules also include the first J region of the first TCR chain and / or the second J region of the second TCR chain. In some embodiments, (i) the first TCR chain is a TCR alpha chain and the second TCR chain is a TCR beta chain, or (ii) the first TCR chain is a TCR gamma chain and the second TCR chain is a TCR delta chain.In some embodiments, TCR V gene is TRAV gene, TRBV gene, TRGV gene or TRDV gene.In some embodiments, the nucleic acid molecule in the second multiple nucleic acid molecule is a double-stranded nucleic acid molecule.In some embodiments, the nucleic acid molecule in the second multiple nucleic acid molecule also includes a sequence encoding a part of a self-cleaving peptide.In some embodiments, the anti-connection sequence is the overhang of the nucleic acid molecule in the second multiple nucleic acid molecule.In some embodiments, the length of the connection sequence or the anti-connection sequence is at least 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,50,60,70,80,90,100,150,200 or more nucleotides. In some embodiments, the method further comprises (i) extending the 3' end of the nucleic acid molecule in the first plurality of nucleic acid molecules that hybridizes to the nucleic acid molecule in the second plurality of nucleic acid molecules and / or (ii) extending the 3' end of the nucleic acid molecule in the second plurality of nucleic acid molecules that hybridizes to the nucleic acid molecule in the first plurality of nucleic acid molecules. In some embodiments, the method further comprises ligating the nucleic acid molecule in the first plurality of nucleic acid molecules to the nucleic acid molecule in the second plurality of nucleic acid molecules.

[0014] In some embodiments, the method described also includes making the nucleic acid molecules comprising the sequence of the first CDR3 and the second CDR3 encoding and the sequence derived from TCR V genes contact with restriction enzyme to produce sticky ends.In some embodiments, the method described also includes making the nucleic acid molecules comprising the sequence of the first CDR3 and the second CDR3 encoding and the sequence derived from TCR V genes contact with the extra nucleic acid molecules comprising the sequence of coding constant region or its part.In some embodiments, the method described also includes the nucleic acid molecules comprising the sequence of the first CDR3 and the second CDR3 encoding and the sequence derived from TCR V genes being connected with extra nucleic acid molecules by sticky ends.In some embodiments, the sequence encoding the first CDR3 and the sequence encoding the second CDR3 are separated by at most about 100,90,80,70,60,50,40,30,20,10 or 5 nucleotides.In some embodiments, the sequence derived from TCR V genes includes the sequence of encoding FR1, CDR1, FR2, CDR2 and FR3. In some embodiments, the sequence derived from a TCR V gene comprises sequences encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2, and FR3.

[0015] On the other hand, the present disclosure provides a composition comprising: a first plurality of nucleic acid molecules, wherein each of the first plurality of nucleic acid molecules comprises a sequence encoding a first CDR3 of a first T cell receptor (TCR) chain and a second CDR3 of a second TCR chain, wherein the first CDR3 and the second CDR3 are from a cognate pair of TCR chains; and a second plurality of nucleic acid molecules, wherein each of the second plurality of nucleic acid molecules comprises a sequence derived from a TCR V gene, and wherein each of the second plurality of nucleic acid molecules does not comprise a sequence encoding the first CDR3 and the second CDR3; wherein (i) each of the first plurality of nucleic acid molecules comprises a sequence encoding a different first CDR3 and / or second CDR3, and / or (ii) each of the second plurality of nucleic acid molecules comprises a sequence derived from a different TCR V gene. In some embodiments, the first plurality of nucleic acid molecules comprises at least about 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 15,000, 20,000, 100,000, 1,000,000, 10,000,000, or more different sequences. In some embodiments, the second plurality of nucleic acid molecules comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or more different TCRs. V gene.

[0016] In some embodiments, each nucleic acid molecule in the first multiple nucleic acid molecules also includes a connecting sequence, wherein a given connecting sequence can be used to connect a given nucleic acid molecule in the first multiple nucleic acid molecules to a given nucleic acid molecule in the second multiple nucleic acid molecules. In some embodiments, each nucleic acid molecule in the second multiple nucleic acid molecules also includes an anti-connection sequence, and the anti-connection sequence is complementary to the connecting sequence. In some embodiments, the connecting sequence is codon-diverse, so that the given connecting sequence of the given nucleic acid molecule in the first multiple nucleic acid molecules is different from other connecting sequences of other nucleic acid molecules in the first multiple nucleic acid molecules. In some embodiments, the connecting sequence encodes an amino acid sequence. In some embodiments, the connecting sequence is in frame with the sequence of the first CDR3 of the first TCR chain and the second CDR3 of the second TCR chain. In some embodiments, the linker sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more nucleotides. In some embodiments, the connecting sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more nucleotides of the TCR V gene adjacent to the sequence encoding the first CDR3 of the first TCR chain or the second CDR3 of the second TCR chain. In some embodiments, the given amino acid sequence encoded by a given connecting sequence is identical or substantially identical to at least one other amino acid sequence encoded by at least one other connecting sequence. In some embodiments, the given amino acid sequence encoded by a given connecting sequence is different from other amino acid sequences encoded by other connecting sequences. In some embodiments, each nucleic acid molecule in the first plurality of nucleic acid molecules also includes the first J district of the first TCR chain and / or the second J district of the second TCR chain. In some embodiments, the composition is a liquid composition. In some embodiments, the first plurality of nucleic acid molecules and the second plurality of nucleic acid molecules are in the same compartment. In some embodiments, the given nucleic acid molecule in the first plurality of nucleic acid molecules is connected to the given nucleic acid molecule in the second plurality of nucleic acid molecules by a given connection sequence. In some embodiments, the given nucleic acid molecule in the first plurality of nucleic acid molecules is hybridized with the given nucleic acid molecule of the second plurality of nucleic acid molecules by a given connection sequence hybridized with a given anti-connection sequence.In some embodiments, the sequence encoding the first CDR3 and the sequence encoding the second CDR3 are separated by at most 100 nucleotides.In some embodiments, the sequence derived from TCR V gene comprises the sequence encoding FR1, CDR1, FR2, CDR2 and FR3.In some embodiments, the sequence derived from TCR V gene comprises the sequence encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and FR3.In some embodiments, every kind of nucleic acid molecule in the first multiple nucleic acid molecule or the second multiple molecule is chemically synthesized.In some embodiments, the length of every kind of nucleic acid molecule in the first multiple nucleic acid molecule is at most about 250,240,230,220,210,200,190,180,170,160,150,140,130,120,110,100 or 50 nucleotides.

[0017] On the other hand, the present disclosure provides a composition comprising a plurality of nucleic acid molecules, each of the plurality of nucleic acid molecules comprising a sequence derived from a T cell receptor (TCR) V gene sequence, wherein the plurality of nucleic acid molecules comprises a first nucleic acid molecule having a first linker sequence and a second nucleic acid molecule having a second linker sequence, wherein the first linker sequence is different from the second linker sequence.

[0018] In some embodiments, every kind of nucleic acid molecule in multiple nucleic acid molecules includes the sequence derived from different TCR V genes.In some embodiments, every kind of nucleic acid molecule in multiple nucleic acid molecules includes different connection sequences.In some embodiments, every kind of nucleic acid molecule in multiple nucleic acid molecules does not include the sequence of the CDR3 encoding TCR chains.In some embodiments, every kind of nucleic acid molecule in multiple nucleic acid molecules does not include the sequence of the constant domain encoding TCR chains.In some embodiments, the sequence derived from TCR V genes includes at least 10,20,30,50,60,70,80,90,100,200,300,400,500,600,700,800,900,1000 or more nucleotides of TCR V genes.In some embodiments, TCR V genes are TRAV genes, TRBV genes, TRGV genes or TRDV genes.

[0019] In another aspect, the present disclosure provides a composition comprising a plurality of nucleic acid molecules, each of the plurality of nucleic acid molecules encoding a CDR3 or a portion thereof of a T cell receptor (TCR) chain, wherein the plurality of nucleic acid molecules comprises a first nucleic acid molecule having a first linking sequence and a second nucleic acid molecule having a second linking sequence, wherein the first linking sequence is different from the second linking sequence.

[0020] In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules also includes the J region of a TCR chain. In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules encodes the first CDR3 of a first TCR chain or its portion and the second CDR3 of a second TCR chain or its portion. In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules also includes the first J region of a first TCR chain and the second J region of a second TCR chain. In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules encodes different CDR3 or its portion of different TCR chains. In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules includes different connection sequences. In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules does not include a TCR V gene greater than 200, 150, 100, 80, 50, 40, 30, 20 or 10 nucleotides. In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules does not include a sequence encoding a constant domain of a TCR chain. In some embodiments, the first connection sequence or the second connection sequence includes a sequence derived from a TCR V gene. In some embodiments, the sequence derived from TCR V gene includes at least 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,50,60,70,80,90,100,150,200 or more nucleotides of the TCR V gene adjacent to the sequence encoding CDR3 in the rearranged gene.In some embodiments, the first connection sequence or the second connection sequence includes a predetermined sequence.In some embodiments, the first connection sequence or the second connection sequence includes a sequence complementary to the TCR V gene sequence.In some embodiments, compositions also includes a second multiple nucleic acid molecule, and each nucleic acid molecule in the second multiple nucleic acid molecule includes a sequence derived from TCR V gene. In some embodiments, the first nucleic acid molecule in the second plurality comprises a first anti-joining sequence that is complementary to the first joining sequence. In some embodiments, the second nucleic acid molecule in the second plurality comprises a second anti-joining sequence that is complementary to the second joining sequence. In some embodiments, the first anti-joining sequence of the first nucleic acid molecule in the second plurality is joined to the first joining sequence of the first nucleic acid molecule in the first plurality. In some embodiments, the second anti-joining sequence of the second nucleic acid molecule in the second plurality is joined to the second joining sequence of the second nucleic acid molecule in the first plurality.

[0021] In another aspect, the present disclosure provides a composition comprising a plurality of nucleic acid molecules, each comprising a sequence encoding at least ten amino acids (e.g., in some cases, encoding at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or more amino acids) of a T cell receptor (TCR) chain, wherein the plurality of nucleic acid molecules comprises a first nucleic acid molecule having a first linker sequence and a second nucleic acid molecule having a second linker sequence, wherein the first linker sequence is different from the second linker sequence, wherein the first linker sequence or the second linker sequence encodes a portion of a TCR chain, and wherein the first linker sequence or the second linker sequence is in frame with a sequence encoding at least ten amino acids (e.g., in some cases, encoding at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or more amino acids) of a TCR chain.

[0022] In some embodiments, the first connection sequence or the second connection sequence comprises at least 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,50,60,70,80,90,100,150,200 or more continuous nucleotides of TCR chain gene, and with the sequence of at least ten amino acids encoding TCR chain in frame.In some embodiments, the first connection sequence and the second connection sequence encode at least two continuous amino acids of TCR chain.In some embodiments, the TCR chain of a part of TCR chain is identical with the TCR chain encoded by the sequence encoding at least ten amino acids.In some embodiments, each nucleic acid molecule in a variety of nucleic acid molecules comprises a sequence derived from TCR V gene. In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules encodes the CDR3 or part thereof of a TCR chain. In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules also includes the J region of a TCR chain. In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules encodes the first CDR3 of a first TCR chain or its part and the second CDR3 or its part of a second TCR chain. In some embodiments, each nucleic acid molecule in a plurality of nucleic acid molecules also includes the first J region of a first TCR chain and the second J region of a second TCR chain. In some embodiments, the sequence encoding the first CDR3 or its part and the sequence encoding the second CDR3 or its part are separated by at most 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 nucleotides. In some embodiments, the first connection sequence or the second connection sequence includes a sequence derived from a TCR V gene. In some embodiments, the first connection sequence or the second connection sequence includes a predetermined sequence. In some embodiments, the first connection sequence includes at least one nucleotide different from the nucleotide of the second connection sequence. In some embodiments, the first connection sequence encodes the same amino acid sequence as the second connection sequence. In some embodiments, the first linker sequence and the second linker sequence encode different amino acid sequences.

[0023] In another aspect, the present disclosure provides a method for producing a plurality of nucleic acid molecules, each nucleic acid molecule in the plurality encoding a T cell receptor (TCR) chain or a region thereof, the method comprising: contacting a first plurality of nucleic acid molecules with a second plurality of nucleic acid molecules to produce a third plurality of nucleic acid molecules, the third plurality of nucleic acid molecules comprising at least two (e.g., at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000 0, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 15,000, 20,000, 100,000, 1,000,000, 10,000,000 or more) different nucleic acid molecules, wherein each of the at least two different nucleic acid molecules has a different sequence encoding a different TCR chain or region thereof, and wherein the at least two different nucleic acid molecules are produced in the same compartment.

[0024] In some embodiments, each nucleic acid molecule in the first plurality of nucleic acid molecules comprises a sequence encoding a CDR3 of a TCR chain. In some embodiments, each nucleic acid molecule in the first plurality of nucleic acid molecules comprises a J region of a TCR chain. In some embodiments, each nucleic acid molecule in the second plurality of nucleic acid molecules comprises a sequence of a TCR V gene derived from a TCR chain. In some embodiments, the TCR V gene is a human TCR V gene. In some embodiments, the TCR V gene is human TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV7, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8-6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV13-2, TRAV1 4, TRAV16, TRAV17, TRAV18, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23, TRAV24, TRAV25, TRAV26-1, TRAV26-2, TRAV27, TRAV29, TRAV30, TRAV34, TRAV35, TRAV36, TRAV38-1, TRAV38-2, TRAV39, TRAV40, or TRAV41. In some embodiments, TCR The V gene is human TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV4-3, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV5-8, TRBV6-1, TRBV6- 2. TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV6-9, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-8 , TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1 or TRBV30. In some embodiments, the sequence derived from the TCR V gene comprises a sequence encoding FR1, CDR1, FR2, CDR2 and FR3.In some embodiments, the sequence derived from the TCR V gene includes the sequence encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and FR3. In some embodiments, TCR chain is TCR alpha chain, TCR beta chain, TCR gamma chain or TCR delta chain. In some embodiments, each nucleic acid molecule in the first multiple nucleic acid molecules also includes the additional sequence of the additional CDR3 encoding additional TCR chains. In some embodiments, each nucleic acid molecule in the first multiple nucleic acid molecules includes the additional J district of additional TCR chains. In some embodiments, TCR chain and additional TCR chain are homologous pairs of TCR chains. In some embodiments, the nucleic acid molecules in multiple nucleic acid molecules encode different TCRs or parts thereof. In some embodiments, the given nucleic acid molecule in the first multiple nucleic acid molecules includes a connecting sequence, which can be used to connect the given nucleic acid molecule in the first multiple nucleic acid molecules to the given nucleic acid molecule in the second multiple nucleic acid molecules. In some embodiments, the given nucleic acid molecule in the first multiple nucleic acid molecules and the given nucleic acid molecule in the second multiple nucleic acid molecules encode functional TCR chains or parts thereof. In some embodiments, a given nucleic acid molecule in the second plurality of nucleic acid molecules comprises an anti-connection sequence, which is complementary to the connection sequence of a given nucleic acid molecule in the first plurality of nucleic acid molecules. In some embodiments, the method further comprises connecting a given nucleic acid molecule in the first plurality of nucleic acid molecules to a given nucleic acid molecule in the second plurality of nucleic acid molecules. In some embodiments, the connection comprises hybridizing a given nucleic acid molecule in the first plurality of nucleic acid molecules to a given nucleic acid molecule in the second plurality of nucleic acid molecules. In some embodiments, hybridization comprises hybridizing the connection sequence of a given nucleic acid molecule in the first plurality of nucleic acid molecules to the anti-connection sequence of a given nucleic acid molecule in the second plurality of nucleic acid molecules. In some embodiments, the method further comprises (i) using a given nucleic acid molecule in the first plurality of nucleic acid molecules as a template to extend the free 3' end of a given nucleic acid molecule in the second plurality of nucleic acid molecules, and / or (ii) using a given nucleic acid molecule in the second plurality of nucleic acid molecules as a template to extend the free 3' end of a nucleic acid molecule in the first plurality of nucleic acid molecules to produce a nucleic acid molecule in a third plurality of nucleic acid molecules. In some embodiments, the method further comprises connecting a given nucleic acid molecule in the first plurality of nucleic acid molecules to a given nucleic acid molecule in the second plurality of nucleic acid molecules. In some embodiments, the method further comprises contacting the nucleic acid molecules in the third plurality of nucleic acid molecules with a restriction enzyme to produce sticky ends. In some embodiments, the method further comprises contacting the nucleic acid molecules in the third plurality of nucleic acid molecules with additional nucleic acid molecules. In some embodiments, the additional nucleic acid molecules encode the constant region or part thereof of the TCR chain. In some embodiments, the method further comprises connecting the nucleic acid molecules in the third plurality of nucleic acid molecules with additional nucleic acid molecules.In some embodiments, at least five (e.g., in some cases, at least about 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 30,000, 40,000 or more) different nucleic acid molecules of the third plurality of nucleic acid molecules are produced in the same compartment. In some embodiments, at least ten different nucleic acid molecules of the third plurality of nucleic acid molecules are produced in the same compartment. In some embodiments, the same compartment is a well, a tube, or a droplet.

[0025] On the other hand, the present disclosure provides a method for producing multiple nucleic acid molecules, comprising: (a) providing a first plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules comprise sequences encoding a first CDR3 of a first T cell receptor (TCR) chain and a second CDR3 of a second TCR chain, wherein the first CDR3 and the second CDR3 are from a homologous pair of TCR chains; (b) providing a second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the second plurality of nucleic acid molecules comprise sequences derived from TCR V genes; and (c) contacting the first plurality of nucleic acid molecules with the second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules are connected to the nucleic acid molecules in the second plurality of nucleic acid molecules to form a linear nucleic acid molecule comprising sequences encoding the first CDR3 and the second CDR3 and sequences derived from the TCR V genes, wherein the sequences encoding the first CDR3 and the second CDR3 and the TCR V genes are derived from a homologous pair of TCR chains. In some embodiments, the first plurality of nucleic acid molecules comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 15,000, 20,000, 100,000, 1,000,000, 10,000,000, or more different sequences. 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 30,

[0026] In another aspect, the present disclosure provides a method for producing a plurality of nucleic acid molecules, comprising: (a) providing a first plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules comprise (i) a synthetic sequence encoding a first CDR3 of a first T cell receptor (TCR) chain and a second CDR3 of a second TCR chain, and (ii) a synthetic sequence encoding a third CDR3 of a third T cell receptor (TCR) chain and a fourth CDR3 of a fourth TCR chain, wherein the first CDR3 and the second CDR3 are from a first homologous pair of TCR chains, and wherein the third CDR3 and the fourth CDR3 are from a second homologous pair of TCR chains; (b) providing a second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the second plurality of nucleic acid molecules comprise a sequence derived from a TCR V gene; and (c) contacting the first plurality of nucleic acid molecules with the second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules are linked to the nucleic acid molecules in the second plurality of nucleic acid molecules to form a nucleic acid molecule comprising a sequence encoding the first CDR3 and the second CDR3 and a sequence derived from the TCR V gene, wherein the sequence encoding the first CDR3 and the second CDR3 and the TCR V genes are derived from cognate pairs of TCR chains. In some embodiments, the first plurality of nucleic acid molecules comprises at least about 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 15,000, 20,000, 100,000, 1,000,000, 10,000,000 or more different sequences. In some embodiments, the second plurality of nucleic acid molecules comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or more different TCRs. V gene.

[0027] On the other hand, the present disclosure provides a method for identifying the sequence of a naturally paired T cell receptor (TCR) in a tissue sample from a subject, comprising: (a) identifying one or more naturally paired TCRs in a sample containing multiple peripheral T cells obtained from the subject One or more paired sequences, wherein each of the one or more paired sequences includes a CDR3 sequence; and (b) identifying the tissue CDR3 sequence of the TCR chain of TCR in the tissue sample, other TCR chains naturally paired with the TCR chain are unknown, wherein the tissue CDR3 sequence matches the CDR3 sequence of at least one paired sequence in one or more paired sequences of one or more naturally paired TCRs, thereby identifying the at least one paired sequence as a naturally paired TCR sequence in the tissue sample. In some embodiments, the identification in (a) includes sequencing one or more naturally paired TCRs in a sample containing multiple peripheral T cells. In some embodiments, sequencing includes single-cell sequencing. In some embodiments, single-cell sequencing includes dividing multiple peripheral T cells into multiple compartments, each compartment containing a single peripheral T cell in multiple peripheral T cells. In some embodiments, the tissue sample is not a body fluid sample. In some embodiments, the tissue sample is a solid tumor sample. In some embodiments, the tissue sample is a fixed or frozen sample. In some embodiments, the sample containing multiple peripheral T cells is a peripheral blood mononuclear cell (PBMC) sample. In some embodiments, the method also includes obtaining a blood sample from the subject before (a). In some embodiments, the method also includes separating peripheral blood mononuclear cells from the blood sample before (a). In some embodiments, the tissue sample comprises tumor infiltrating T cells.

[0028] On the other hand, the present disclosure provides a method for identifying a target reactive T cell receptor (TCR), comprising: (a) providing a cell comprising a TCR identified using the methods described herein; and (b) contacting the cell with a target antigen presented by an antigen presenting cell (APC), wherein the cell binds to the target antigen presented by the APC through the TCR, thereby identifying the TCR as a target reactive TCR. In some embodiments, the target antigen is a tumor antigen (e.g., a tumor-associated antigen or a tumor-specific antigen). In some embodiments, the method further comprises delivering a sequence encoding the target reactive TCR into a host cell. In some embodiments, the method further comprises administering the host cell to a subject. In some embodiments, the host cell is a T cell. In some embodiments, the T cell is an autologous T cell. In some embodiments, the T cell is an allogeneic T cell. In some embodiments, the cell is a reporter cell line, and the reporter cell line comprises a reporter gene expressed when the cell binds to the target antigen presented by the APC.

[0029] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only exemplary embodiments of the present disclosure are shown and described. It should be appreciated that the present disclosure can have other and different embodiments, and that several details thereof can be modified in various aspects, all without departing from the disclosure. Therefore, the drawings and description should be regarded as illustrative rather than restrictive in nature.

[0030] Incorporated by Reference

[0031] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and the accompanying drawings (also referred to herein as "Figures", "Figs" and "FIGUREs") which illustrate exemplary embodiments in which the principles of the present invention are utilized, wherein:

[0033] Figure 1A-Figure 1C An exemplary scheme for generating a nucleic acid construct encoding a T cell receptor is depicted.

[0034] Figure 2A Depicted are exemplary simulation results using the methods described herein.

[0035] Figure 2B Depicted are exemplary simulation results using the methods described herein.

[0036] Figure 3A Depicted are exemplary simulation results using the methods described herein.

[0037] Figure 3B Depicted are exemplary simulation results using the methods described herein.

[0038] Figure 4A Schematic representation of the germline genomic DNA of the TCR V genes.

[0039] Figure 4B Schematic representation of the rearranged genomic DNA depicting the TCR VJ genes.

[0040] Figure 4C Schematic representation of the rearranged genomic DNA depicting the TCR VDJ genes.

[0041] Figure 5 A scheme is depicted of potential challenges associated with joining a CDR3-J polynucleotide to a correct V gene germline polynucleotide. The dashed arrows depict that a connection may occur between a CDR3-J polynucleotide and an incorrect V gene germline polynucleotide.

[0042] Figure 6 A scheme for connecting a CDR3-J polynucleotide (grey box connected to a white box) to a specified, presynthesized V gene germline polynucleotide (black box connected to a grey box indicated by a thin arrow) by overlapping primer extension is depicted. The top thick arrow (603) depicts hybridization between a connecting sequence (601) on a presynthesized V gene germline polynucleotide and a connecting sequence (602) on a CDR3-J polynucleotide. The bottom thick arrow (604) depicts primer extension. 601 can be referred to as a connecting sequence, and 602 can be referred to as an anti-connection sequence (or vice versa).

[0043] Figure 7 Depicted is the joining of a CDR3-J polynucleotide to a specified V gene germline polynucleotide using an arbitrary joining sequence (701) and an anti-joining sequence (702).

[0044] Figure 8 The general principle of TCR gene self-assembly is depicted. 801: Presynthesized V gene germline polynucleotide. 802: Polynucleotide comprising a CDR3-J sequence (e.g., a CDR3-J polynucleotide). 803: Nucleic acid sequence comprising a V gene germline polynucleotide sequence and a CDR3-J sequence. X is the number of polynucleotides that are each part of a different V gene germline polynucleotide. Y is the number of CDR3-J polynucleotides. Y can be much greater than X. Arrows represent batch reactions in which each CDR3-J polynucleotide is linked to a specified, presynthesized V gene germline polynucleotide.

[0045] Fig. 9A Depicted is an example workflow for using blood samples to identify tumor-infiltrating TCRs in tumor samples.

[0046] Fig. 9B Describes the use Fig. 9A Exemplary applications of TCRs identified using the methods shown.

[0047] Fig. 9C Describes the use Fig. 9A Exemplary applications of TCRs identified using the methods shown.

[0048] Fig. 10A Depicted are exemplary simulation results using the methods described herein.

[0049] Fig. 10B Depicted are exemplary simulation results using the methods described herein.

[0050] Fig.11A Depicted are exemplary simulation results using the methods described herein.

[0051] Fig. 11B Depicted are exemplary simulation results using the methods described herein.

[0052] Fig.12 Depicted are exemplary next generation sequencing data for evaluating the gene assembly methods described herein.

[0053] Fig.13 Depicted are exemplary next generation sequencing data for evaluating the gene assembly methods described herein.

[0054] Fig.14 Depicted are exemplary next generation sequencing data for evaluating the gene assembly methods described herein.

[0055] Fig.15 Depicted are exemplary next generation sequencing data for evaluating the gene assembly methods described herein.

[0056] Fig.16 Depicted are exemplary next generation sequencing data for evaluating the gene assembly methods described herein.

[0057] Fig.17 Depicted are exemplary next generation sequencing data for evaluating the gene assembly methods described herein.

[0058] Fig.18 Depicted are exemplary next generation sequencing data for evaluating the gene assembly methods described herein. DETAILED DESCRIPTION

[0059] In this disclosure, the use of the singular includes the plural unless specifically stated otherwise. Likewise, the use of "or" means "and / or" unless otherwise stated. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are not intended to be limiting.

[0060] The term "about" or "approximately" means that a particular value determined by one of ordinary skill in the art is within an acceptable error range, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, such as within 5 times, or within 2 times. When specific values ​​are described in the application and claims, unless otherwise stated, it should be assumed that the term "about" means within an acceptable error range for the specific value.

[0061] When the term "at least", "greater than", or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least", "greater than", or "greater than or equal to" applies to each value in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0062] When the term "not greater than," "less than," or "less than or equal to" precedes the first value in a series of two or more values, the term "not greater than," "less than," or "less than or equal to" applies to each value in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0063] The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably in the present disclosure. They can refer to polymeric forms of nucleotides of various lengths. They can contain deoxyribonucleotides and / or ribonucleotides or their analogs. Polynucleotides can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) or variants thereof. Nucleotides can include nucleosides and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate (PO3) groups. Nucleotides can include a nucleobase, a pentose (ribose or deoxyribose) and one or more phosphate groups. Polynucleotides can have any three-dimensional structure and can perform various functions. Polynucleotides can have various configurations, such as linear, circular, stem-loop and branched. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), circular RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The polynucleotide may comprise one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs and / or modified nucleotides.

[0064] As used herein, the term "sequence" refers to the order of nucleotides in a nucleic acid molecule, or the order of amino acid residues of a peptide. A nucleic acid sequence may be a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence; may be linear, circular or branched; and may be single-stranded or double-stranded. A sequence may be mutated so that it is different from a reference sequence (e.g., a wild-type sequence). A sequence may have any length, such as a length between 2 and 1,000,000 or more amino acids or nucleotides (or any integer value therebetween or thereon), such as between about 100 and about 10,000 nucleotides or between about 200 and about 500 amino acids or nucleotides. In some cases, a given nucleic acid sequence may encompass the sequence information of a given nucleic acid sequence and the reverse complementary sequence of a given nucleic acid sequence. In some cases, a DNA sequence may encompass the sequence information of a corresponding RNA sequence transcribed by DNA. A sequence may be an alphabetic representation of a polynucleotide or polypeptide molecule. A sequence may be a piece of information that can be used by a computer processor. In some cases, a nucleic acid sequence may be used to refer to the physical nucleic acid molecule itself.

[0065] As used herein, the term "blunt end" refers to the end of a double-stranded nucleic acid molecule in which substantially all of the nucleotides at the end of one strand of the nucleic acid molecule are base-paired with the opposite nucleotides in the other strand of the same nucleic acid molecule. If the end of a nucleic acid molecule includes a single-stranded portion of at least one nucleotide in length, the nucleic acid molecule is not blunt-ended, referred to herein as an "overhang" or "sticky end."

[0066] As used herein, the term "TCR V gene" refers to the genomic nucleic acid sequence of the variable (V) gene of the T cell receptor of germline configuration, which comprises a sequence (such as L-PART1 defined in IMGT), an intron (such as V-INTRON defined in IMGT) and an exon (such as V-EXON defined in IMGT) encoding the first part of the leader peptide, with 5'UTR and 3'UTR (including recombination signal sequence). The recombination signal sequence may include a heptamer (such as V-HEPTAMER defined in IMGT) and a nonamer (such as V-NONAMER defined by IMGT) separated by a spacer element (such as V-SPACER defined by IMGT). V-EXON covers the sequence of the second part (L-PART2) and V-REGION encoding the leader peptide. The example of TCR V gene includes TCR α variable (TRAV) gene, TCR β variable (TRBV) gene, TCR γ variable (TRGV) gene and TCR δ variable (TRDV) gene. Nucleic acid as described herein may include a sequence derived from a TCR V gene. "Derived from" means a sequence with at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or 100% sequence identity to a reference sequence. The sequence derived from a TCR V gene can be the full-length sequence of the genomic nucleic acid sequence of a TCR V gene as described above. The sequence derived from a TCR V gene can be a part of a TCR V gene, comprising at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more nucleotides of a TCR V gene. The sequence derived from a TCR V gene can be a codon-optimized (or codon-diverse) nucleic acid sequence. The codon-optimized sequence of a given nucleic acid sequence refers to a modified nucleic acid sequence, and its protein coding region encodes the same amino acid sequence as the protein coding region of a given nucleic acid. The modified nucleic acid sequence may have a sequence different from a given nucleic acid sequence or may be derived from a given nucleic acid. Codon optimization can be implemented to remove restriction sites, remove unwanted secondary structures in the polynucleotide sequence, promote the correct connection of the specified presynthesized parts of the CDR3-J polynucleotide and the TCR V gene, or for other purposes. Codon optimization or codon diversification can be achieved by changing one or more nucleotides of a given nucleic acid sequence. For example, codon optimization or codon diversification can be achieved by computational methods. Codon optimization and codon diversification are used interchangeably in the present disclosure.

[0067] As used herein, the term "V-REGION" refers to the coding region of a TCR V gene (including 1 or 2 nucleotides preceding the V-HEPTAMER, if present) in germline genomic DNA or cDNA, or the variable (V) region that is typically trimmed at the 3' end by V-(D)-J rearrangement in rearranged genomic DNA or cDNA.

[0068] As used herein, the term "D-REGION" refers to the coding region of a TCR D gene (including 1 or 2 nucleotides after the 5' D-HEPTAMER and / or before the 3' D-HEPTAMER, if present) in germline genomic DNA or cDNA, or the diversity (D) region that is typically trimmed at the 5' and / or 3' by DJ or VDJ rearrangement in partially rearranged or rearranged genomic DNA or cDNA.

[0069] As used herein, the term "J-REGION" refers to the coding region of a TCR J gene (including 1 or 2 nucleotides following the J-HEPTAMER, if present) in germline genomic DNA or cDNA, or the joining (J) region that is typically trimmed at the 5' end by V-(D)-J rearrangement in rearranged genomic DNA or cDNA.

[0070] As used herein, the term "VJ-REGION" refers to the coding region of the TCR chain containing V-REGION and J-REGION in the rearranged genomic DNA or cDNA.

[0071] As used herein, the term "VDJ-REGION" refers to the coding region of the TCR chain comprising V-REGION, D-REGION and J-REGION in the rearranged genomic DNA or cDNA.

[0072] The terms "link" or "connect" are used interchangeably in this disclosure. They refer to physically connecting two or more nucleic acid molecules. Two or more nucleic acid molecules can be connected so that the two or more nucleic acid molecules form a continuous nucleic acid molecule. Two or more nucleic acid molecules can be covalently linked or non-covalently linked. Linking can be achieved in a variety of ways, including the formation of hydrogen bonds, ionic bonds, and covalent bonds or van der Waals forces.

[0073] The percentage (%) of sequence identity relative to a reference nucleic acid sequence (or peptide sequence) is the percentage of nucleotides (or amino acid residues in the case of a peptide sequence) identical to the nucleotides (or amino acid residues) in the candidate sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage of sequence identity can be achieved in various ways within the technical scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, CLUSTALW, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0074] The term "substantially identical" and its grammatical equivalents as applied to nucleic acid or amino acid sequences refers to a nucleic acid or amino acid sequence comprising a sequence having at least 90% or more, at least 95%, at least 98%, or at least 99% sequence identity compared to a reference sequence using the above-mentioned programs (e.g., BLAST) using standard parameters. For example, the default values ​​used by the BLASTN program (for nucleotide sequences) are a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the default values ​​used by the BLASTP program are a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)).

[0075] Overview

[0076] High-throughput, paired sequencing can be used to sequence T cell receptor (TCR).For example, with the development of single cell technology, single T cell can be assigned to isolated compartments, wherein TCR α and β chain mRNA from the same T cell can be attached to the same unique barcode.Some of these systems are commercially available (for example, by 10X Genomics).Record T cell receptor α variable (TRAV) gene identity, CDR3α sequence, T cell receptor α connection (TRAJ) gene identity, T cell receptor β variable (TRBV) gene identity, CDR3β sequence and T cell receptor β connection (TRBJ) gene identity pairing sequence information can allow reconstruction of full length, expressible TCR.However, the technology of synthesizing such TCR sequences in the form of DNA or RNA that can be introduced into cells for functional studies or screening may be low-throughput.The present disclosure provides a variety of methods and compositions, which can allow ultra-high throughput to construct polynucleotides encoding TCR sequences (for example, in some cases, paired, full-length, expressible TCR sequences).

[0077] T cell receptor (TCR)

[0078] TCR can be used to give T cells the ability to recognize antigens associated with various cancers or infectious organisms. TCR is composed of two chains, such as α chain and β chain or γ chain and δ chain. The proteins constituting these chains are encoded by DNA, and the DNA uses a unique mechanism to produce the huge diversity of TCR. This multi-subunit immune recognition receptor associates with the CD3 complex and binds to the peptides presented by MHC class I and class II proteins on the surface of antigen presenting cells (APC). The combination of TCR with the antigen peptide on APC can be a central event in T cell activation, and the combination occurs at the immune synapse of the T cell and APC contact point.

[0079] In the context of MHC class I molecules, TCRs can recognize T cell epitopes. MHC class I proteins can be expressed in all nucleated cells of higher vertebrates. MHC class I molecules are heterodimers consisting of a 46-kDa heavy chain non-covalently associated with a 12-kDa light chain beta-2 microglobulin. In humans, there are several MHC alleles, such as HLA-A2, HLA-Al, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45, and HLA-Cw8. In some embodiments, the MHC class I allele is an HLA-A2 allele, which is expressed by approximately 50% of the population in some populations. In some embodiments, the HLA-A2 allele can be an HLA-A*0201, *0202, *0203, *0206, or *0207 gene product. In some cases, there may be differences in subtype frequencies between different populations. For example, in some embodiments, more than 95% of the HLA-A2 positive Caucasian population is HLA-A*0201, while in the Chinese population, the frequencies are reported to be approximately 23% HLA-A*0201, 45% HLA-A*0207, 8% HLA-A*0206, and 23% HLA-A*0203.

[0080] In some embodiments, TCR can recognize T cell epitopes in the context of MHC class II molecules. MHC class II proteins can be expressed in a subset of APCs. In humans, there are several MHC class II alleles, such as DR1, DR3, DR4, DR7, DR52, DQ1, DQ2, DQ4, DQ8 and DPI. In some embodiments, the MHC class II allele is HLA-DRB1*0101, HLA-DRB*0301, HLA-DRB*0701, HLA-DRB*0401 or HLA-DQB1*0201 gene product.

[0081] The TCR chain may include a variable domain (or variable region) and a constant domain (or constant region). The variable domain may be a V-DOMAIN defined by the IMGT unique numbering system. The variable domain may correspond to the VJ-REGION or VDJ-REGION of the TCR chain. The constant domain may be a C-DOMAIN defined by the IMGT unique numbering system. In some cases, the constant domain may be a part of the constant region. For example, the full-length constant region may include a constant domain (extracellular region), a connecting region, a transmembrane region, and a cytoplasmic region.

[0082] The variable domains of TCRα or TCRδ chains can be encoded by multiple variable (V) and connecting (J) gene segments in the germline, while the variable domains of TCRβ or TCRγ chains are additionally encoded by diversity (D) gene segments. Each gene segment can be flanked by a recombination signal sequence. The recombination signal can include heptamers and nonamers separated by spacer elements. The length of the spacer element can be 12 or 23bp. During V(D)J recombination, one random allele of each gene segment is recombined with other alleles to form a functional variable domain. The recombination of variable domains with constant (C) gene segments can produce functional TCR chain transcripts. In addition, random nucleotides can be added and / or deleted at the connection sites between gene segments. This process can lead to strong combinations (depending on which gene regions will be recombined) and connection diversity (depending on which and how many nucleotides are added / deleted), thereby producing a large number of highly variable TCR libraries, which can ensure that a large number of antigens are recognized. Additional diversity can be achieved by pairing α and β or γ and δ chains (also referred to as "assembly") to form functional TCRs. Through recombination, random insertion, deletion and substitution, a small set of genes encoding T cell receptors has the potential to generate 10 15 with 10 20 The potential of a TCR clonotype. As used herein, "clonotype" refers to a population of immune cells carrying the same immune receptor. For example, a clonotype refers to a population of T cells carrying the same TCR, or a population of B cells carrying the same BCR (or antibody). In the context of immune receptor diversity, "diversity" refers to the number of clonotypes of immune receptors (e.g., TCR, BCR, and antibodies) in a population. As used herein, "homologous pair combination" refers to a natural combination of two chains (e.g., TCRα and TCRβ, or TCRγ and TCRδ) of a TCR from a T cell. The same homologous pair combination of two chains can produce the same TCR. For example, a T cell with the same clonotype has the same homologous pair combination of TCRα and TCRβ chains. A higher diversity in a clonotype can indicate a higher diversity in a homologous pair combination.

[0083] Each TCR chain may contain three hypervariable loops in its structure, referred to as complementary determining regions (CDR1-3). CDR1 and CDR2 may be encoded by V genes and may be required for the interaction of TCR with the MHC complex. However, CDR3 is partially encoded by (1) the junction region between V and J genes (in the case of TCRα or TCRγ), or (2) the junction region between V and D genes and the junction region between D and J genes (in the case of TCRβ or TCRδ), and may therefore be highly variable. CDR3 may be the region of the TCR that is in direct contact with the peptide antigen. CDR3 may be used as a target region for determining T cell clonotypes. The sum of all TCRs of an individual's T cells is referred to as a TCR library or TCR repertoire. The TCR library may change with the onset and progression of the disease. Therefore, determining the immune library status under different disease states (such as cancer, autoimmune diseases, inflammatory diseases, and infectious diseases) can be used for disease diagnosis and prognosis.

[0084] TCR should be understood to cover full-length TCR and its antigen binding portion or antigen binding fragment (also referred to as MHC-peptide binding fragment). In some embodiments, TCR is a complete or full-length TCR. In some embodiments, TCR is an antigen binding portion, which is smaller than the full-length TCR, but binds to a specific antigen peptide that binds to an MHC molecule (e.g., an MHC-peptide complex). In some cases, the antigen binding portion or fragment of TCR may contain only a portion of the domain of the full-length or complete TCR, but is still able to bind to the epitope (e.g., MHC-peptide complex) bound by the complete TCR. In some cases, the antigen binding portion or fragment of TCR contains a variable domain of TCR, such as a variable α chain and a variable β chain of TCR, which is sufficient to form a binding site for binding to a specific MHC-peptide complex, such as usually in the case of each chain containing three complementary determining regions. Including a polypeptide or protein having a binding domain as an antigen binding domain or homologous to an antigen binding domain.

[0085] TCR molecules can be formed by α chains (α chains or TCRα chains, encoded by TRA genes / sequences) and β chains (β chains or TCRβ chains, encoded by TRB genes / sequences), or γ chains (γ chains or TCRγ chains, encoded by TRG genes / sequences) and δ chains (δ chains or TCRδ chains, encoded by TRD genes / sequences). These immune receptor chains can have variable domains (e.g., encoded by rearranged VDJ or VJ regions). Portions of the variable domains can be highly variable. Hypervariable regions may include complementary determining regions (CDRs), such as CDR1, CDR2, and CDR3. In some cases, in a T cell, only one functional α chain sequence and one functional β chain sequence may be expressed. In some cases, in a T cell, only one functional γ chain sequence and one functional δ chain sequence may be expressed.

[0086] Chip-based oligonucleotide synthesis: opportunities and challenges

[0087] Although the high-throughput oligonucleotide synthesis technology based on the chip may have developed to the extent that hundreds of thousands or even millions of oligonucleotides with arbitrary sequences can be synthesized at one time, the length of the oligonucleotide synthesized in this way can be limited to about 200 to 300 bases long. In contrast, the length of the full-length TCR construct can be nearly two thousand bases. At first glance, the synthesis based on the chip seems to be insufficient to solve the TCR gene synthesis problem. However, the inspection of the TCR structure can reveal opportunities. First, the constant region (for example, TRAC and TRBC) of the TCR α chain and the β chain can be constant. Therefore, the polynucleotide sequence encoding the constant region of the TCR chain can be attached to the rest of the TCR sequence. Secondly, unlike the BCR / antibody sequence, TCR may not undergo somatic hypermutation, which means that the sequence outside the CDR3 region can be germline derived. Therefore, the polynucleotides each comprising a sequence derived from a TCR V gene or a part thereof can be pre-synthesized. The sequence derived from the TCR V gene can be a part of the TCR V gene. The sequence derived from the TCR V gene can be a codon-optimized sequence or a nucleotide comprising one or more modifications. For example, the sequence derived from TCR V gene (called LV-REGION) of the coding sequence including L-PART1 (the first part of leader peptide), L-PART2 (the second part of leader peptide), FR1, CDR1, FR2, CDR2 and FR3 can be pre-synthesized. For another example, the sequence derived from TCR V gene (called V-REGION) of the coding sequence including FR1, CDR1, FR2, CDR2 and FR3 can be pre-synthesized. The nucleic acid sequence segment of L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 or FR3 can be defined according to the IMGT unique numbering system (http: / / www.imgt.org). In some cases, the sequence derived from TCR V gene can include the sequence starting from the sequence of encoding L-PART1 and terminating at the codon encoding the second conserved cysteine ​​(for example, the 2nd CYS defined by IMGT, corresponding to the codon of the conserved cysteine ​​at the position 104 of V-DOMAIN). Since there are about 80 or more TCR V genes (e.g., TRAV and TRBV genes) in the human genome, such a "V gene germline polynucleotide library" (e.g., Figure 8, 801 and bracket X) is feasible. In some cases, a subset of the TCR V genes of a species (e.g., human or mouse) is synthesized to produce a "V gene germline polynucleotide library". All identified TCR V genes or a subset of TCRV genes can be synthesized. For example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or more TCRs of a species can be synthesized. In some cases, the TCR V genes of all identifications of the synthetic species are used to produce libraries. The TCR V gene can be TRAV, TRBV, TRGV or TRDV. As described herein, in some cases, "V gene germline polynucleotides" refers to a part of the genome or codon-optimized polynucleotides of the TCR V gene. The sequence derived from the TCR V gene can be a V gene germline polynucleotide. The length of the sequence between FR3 and the constant region (for example, CDR3 plus the remainder of the J region, referred to herein as "CDR3-J") can be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more nucleotides, or in some cases, the length can be up to about 90 nucleotides. The length of the CDR3-J sequence of the α chain and β chain of TCR can be at least about 50, 60, 70, 80, 90, 100, 120, 150, 180 or more nucleotides. The CDR3-J sequences of the α and β chains of a TCR (in some cases, up to about 180 nucleotides in total length) can be included in an oligonucleotide (referred to as a "paired CDR3-J oligomer," "paired CDR3-J oligonucleotide," or "paired CDR3-J polynucleotide," which can be used interchangeably) that can be subjected to chip-based synthesis (e.g., Figure 8, 802 and brackets Y covering 802). In some cases, the paired CDR3-J polynucleotides may include the CDR3-J sequences of TCRγ chains and the CDR3-J polynucleotides of TCRδ chains. As used herein, the terms "CDR3-J polynucleotides", "CDR3-J oligonucleotides" and "CDR3-J oligomers" (which are used interchangeably) refer to polynucleotide sequences comprising one or more CDR3-J sequences. CDR3-J polynucleotides may be paired CDR3-J polynucleotides (e.g., comprising CDR3-J sequences from paired TCR chains). CDR3-J polynucleotides (e.g., non-paired) may contain only the CDR3-J sequences from one of the paired TCR chains. For example, CDR3-J polynucleotides may contain only the CDR3-J sequences from TCRα chains, TCRβ chains, TCRγ chains or TCRδ chains. A remaining challenge may be to convert such paired CDR3-J oligonucleotides into expressible TCR constructs at high throughput (e.g., constructing >1,000 TCRs in one batch). Using the methods described herein, paired CDR3-J oligonucleotides can be ligated to their corresponding V gene germline polynucleotides in a batch reaction (e.g., Figure 8, 803). In some cases, a library of CDR3-J polynucleotides (e.g., paired or unpaired) may comprise at least about 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 15,000, 20,000, 100,000, 1,000,000, 10,000,000 or more different sequences. The V gene germline polynucleotide library can comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or more TCRs V gene.When using the method described herein, a plurality of at least 2,5,10,20,50,100,200,300,400,500,1,000,1,500,2,000,2,500,3,000,3,500,4,000,5,000,6,000,7,000,8,000,9,000,10,000,12,000,15,000,20,000,100,000,1,000,000,10,000,000 or more different sequences of the TCR of different natural pairings of encoding can be produced in batches in a single compartment.The TCR of natural pairing can be produced in batches in a single compartment.

[0088] Examples of germline or rearranged gene constructs of nucleic acid molecules comprising TCR V gene sequences are described in Figure 4A-4C For example, Figure 4A The germline genomic DNA of the TCR V gene is shown, which contains L-PART1, V-INTRON, V-EXON and recombination signal sequences (V-HEPTAMER, V-SPACER and V-NONAMER). Figure 4A Two conserved cysteines are also shown in FIG. After V-(D)-J recombination, an exemplary construct of rearranged genomic DNA is shown in FIG. Figure 4B or Figure 4CCDR3 may be encoded by (i) the junction (or junction region) between V-REGION and J-REGION or (ii) the junction between V-REGION and D-REGION and the junction between D-REGION and J-REGION.

[0089] TCR V genes can be very diverse. In humans, more than 40 functional V genes of TRA have been identified, including, for example, TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV7, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8-6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV14-2, TRAV15-3, TRAV16-4, TRAV17-5, TRAV18-6, TRAV19-1, TRAV20-2, TRAV21-3, TRAV22-4, TRAV23-6, TRAV24-1, TRAV25-2, TRAV26-3, TRAV27-4, TRAV28-6, TRAV29-1, TRAV29-2, TRAV30-4, TRAV31-5, TRAV32-6, TRAV33-1, TRAV34-2, TRAV35-3, TRAV36-4, TRAV37-5, TRAV38-6, TRAV39-1, TRAV40-2, TRAV41-1, TRAV42-2, TRAV43-3, TRAV44-4, TRAV45-5 In some embodiments, TRAV1-1 and TRAV1-2 are from the same subgroup.As used herein, " group " is to share the same " genotype " (for example, V, D, J or C type) and potentially participate in the synthesis of the polypeptide of same " chain type ". By extension, a group includes related pseudogenes and orphons. "Subgroup" means a set of genes that belong to the same group in a given species and share at least 75% identity at the nucleotide level (in the germline configuration of V, D and J).

[0090] In humans, more than 40 functional V genes of TRB have been identified, including, for example, TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV4-3, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV5-8, TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV6-9, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-8, TRBV7-9, TRBV8-1, TRBV8-2, TRBV8-3, TRBV8-4, TRBV8-5, TRBV8-6, TRBV8-8, TRBV8-9, TRBV9-1, TRBV9-2, TRBV9-3, TRBV9-4, TRBV9-5, TRBV9-6, TRBV9-8, TRBV9-9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV10-4, TRBV10-5, TRBV10-6, TRBV10-8, TRBV10-9, TRBV11-2, TRBV11-3, TRBV11-4, TRBV11-5, TRBV11-6, TRBV11-8, TRBV11-9, TRBV12-1 TRBV7-7, TRBV7-8, TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1, and TRBV30. V genes of other species (e.g., mouse) can be found in the IMGT database.

[0091] Diversify the connection sequence

[0092] Joining the V gene germline polynucleotide to the CDR3-J polynucleotide can be accomplished by molecular biology techniques such as ligation and overlapping primer extension ( Figure 6 However, to fully exploit the power of chip-based oligonucleotide synthesis, thousands or more CDR3-J oligonucleotides can be ligated to their corresponding V gene germline polynucleotides in a batch reaction (e.g. Figure 8 The main challenge in doing this can be the V gene germline polynucleotide ( Figure 6 , 601) and CDR3-J ( Figure 6 , 602) can be a conserved FR3 region. Therefore, in a batch reaction, it may be difficult to control which V gene germline polynucleotide is connected to which CDR3-J (e.g. Figure 5As shown, where the solid arrow represents connection to the correct V gene germline polynucleotide, and the dotted arrow represents connection to an incorrect V gene germline polynucleotide). For example, the TCR sequence can be formed by TRBV4-1 connected to a specific CDR3-Jβ sequence. In a batch reaction, there may be V gene germline polynucleotides of TRBV4-1 and TRBV4-2, and the FR3 regions of these TRBV genes may be highly similar. Therefore, the CDR3-J oligonucleotide of this TCR may be incorrectly connected to the TRBV4-2 germline polynucleotide. In order to alleviate this problem, codon diversification can be used to produce differences between different FR3 sequences. For example, the connection sequence can be codon-diverse so that they can have different nucleic acid sequences, even if they can encode the same amino acid sequence. Codon diversification can be achieved by computational methods (such as the method shown in Example 2). Multiple nucleic acid sequences can be generated by assigning codons to amino acids randomly or according to arbitrary rules, wherein each nucleic acid sequence can encode the same amino acid sequence. Then, multiple nucleic acid sequences can be evaluated by calculation according to arbitrary rules to assign scores. Arbitrary rules may consider factors such as restriction sites, the tendency of hybridizing with unwanted sequences, the tendency of hybridizing with given sequences or the secondary structure that is not needed in the sequence.Then, according to the score, from the multiple nucleic acid sequences, a nucleotide sequence may be selected as the ligated sequence of the diverse codons.The ligated sequence of the diverse codons may be used to realize the correct connection of the specified presynthesized part of CDR3-J polynucleotide and TCR V gene.In the "V gene germline polynucleotide library" comprising some or all of known TCR V genes, for example, each different TCR V gene may have different ligated sequences, which may be used to correctly connect with corresponding CDR3-J oligonucleotide to form TCR chains according to reference sequences.The reference sequence may be produced by sequencing the homology of the TCR chains.However, in some cases, it may be unclear that the ligated sequence can be diversified, and the connection between the V gene germline polynucleotide and the CDR3-J oligonucleotide can be correct in batch reaction.As shown in Example 2, the FR3 district of human TCR V gene may be diversified, so that in fact the "wrong connection probability" of any given CDR3-J sequence cannot be detected. The algorithm listed in Example 2 can be used to generate "codon-diversified V gene germline polynucleotides" and their corresponding CDR3-J sequences.

[0093] Once a diverse set of connection sequences is found, many methods using molecular biology techniques (e.g., ligation, restriction digestion, cyclization) can be used to convert the CDR3-J oligonucleotide library into a full-length, expressible TCR library. Example 1 provides an exemplary workflow. The method provided herein can also be used to generate a library of a single TCR chain (e.g., a non-paired chain) in a batch reaction. For example, in order to generate a library of a TCR α chain, each individual CDR3-J oligonucleotide may include a CDR3 and a J region from a TCR α chain, but may not include another CDR3 and a J region from a TCR β chain, and then the CDR3-J oligonucleotide can be used to connect to the corresponding TRAV gene to form a TCR α chain.

[0094] Methods for constructing nucleic acid molecules encoding TCRs

[0095] The nucleic acid molecules encoding TCR described herein can be constructed from two or more nucleic acid fragments. In some embodiments, the two or more nucleic acid fragments can be referred to as the first nucleic acid molecule, the second nucleic acid molecule, the third nucleic acid molecule, the fourth nucleic acid molecule, etc. When constructing nucleic acid molecules, standard molecular biology techniques can be used, including but not limited to hybridization, extension, connection and enzyme digestion / cutting.

[0096] Nucleic acid fragments as described herein can encode TCR chains or parts thereof.For example, the part of the TCR chain encoded by the nucleic acid fragment may include more than or equal to about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250 or more amino acids.Nucleic acid fragments may include sequences encoding functional TCR chains.Functional TCR chains may be or may not be full-length TCR chains.Functional TCR chains may include one or more mutations or modifications.In some cases, functional TCR chains may be incorporated into TCR complexes (for example, with TCR α, TCR β, CD3 γ, CD3 δ, CD3 ε and ζ chains) when expressed in host cells.In some cases, functional TCR may be combined with its target ligand.In some cases, functional TCR may be incorporated into cell membranes when expressed in host cells. In some cases, a functional TCR can be expressed in a host cell.

[0097] The nucleic acid fragment used to construct a nucleic acid molecule encoding a TCR or a portion thereof may comprise a sequence encoding CDR3.

[0098] The nucleic acid fragment for constructing a nucleic acid molecule encoding a TCR or a portion thereof may include a sequence encoding a first CDR3 of a first TCR chain and a second CDR3 of a second TCR chain, wherein the first CDR3 and the second CDR3 are derived from a homologous pair of TCR chains. In some embodiments, the sequence encoding the first CDR3 and the sequence encoding the second CDR3 are separated by at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 nucleotides.

[0099] The nucleic acid fragment for building the nucleic acid molecule of coding TCR or its part can comprise TCR V gene sequence or its part.The nucleic acid fragment for building the nucleic acid molecule of coding TCR or its part can comprise the sequence derived from TCR V gene sequence.The sequence derived from TCR V gene can comprise V-REGION nucleotide sequence.The sequence derived from TCR V gene can comprise the sequence of coding FR1, CDR1, FR2, CDR2 and / or FR3 nucleotide sequence.The sequence derived from TCR V gene can comprise the sequence of coding leader peptide.The sequence derived from TCR V gene can comprise the sequence of coding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and / or FR3 nucleotide sequence.The sequence derived from TCR V gene can comprise or can be a part of TCR V gene.The length of the part of TCRV gene can be at least 10 nucleotides. For example, the length of the part of TCR V gene can be greater than or equal to about 10, 20, 30, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides. The sequence derived from the TCR V gene can include one or more modified nucleotides. The sequence derived from the TCR V gene can be codon optimized (or codon diversified) so that it has a sequence different from the TCR V gene or its part, but it can encode the same amino acid sequence. The sequence derived from the TCR V gene may not include the sequence of a part for encoding CDR3. The sequence derived from the TCR V gene may not include the sequence of the junction area of ​​the rearranged gene.

[0100] The nucleic acid fragment for constructing the nucleic acid molecule encoding TCR or its part may include the sequence encoding constant domain or its part. The nucleic acid fragment for constructing the nucleic acid molecule encoding TCR or its part may include the sequence encoding constant region or its part. In some cases, constant domain or constant region is TCR alpha constant domain or constant region, TCR beta constant domain or constant region, TCR gamma constant domain or constant region or TCR delta constant domain or constant region. In some cases, constant region includes constant domain. In some cases, constant region also includes transmembrane region, connection region, cytoplasmic region or its combination.

[0101] The nucleic acid fragment for constructing a nucleic acid molecule encoding TCR or part thereof may include a connection sequence. A connection sequence can be used to connect a nucleic acid molecule to another nucleic acid molecule. A connection sequence of a nucleic acid molecule can hybridize with an anti-connection sequence of another nucleic acid molecule (e.g., to form one or more base pairs). An anti-connection sequence can be complementary to a connection sequence (e.g., completely or substantially complementary). An anti-connection sequence can hybridize with a connection sequence under certain conditions (e.g., temperature, buffer conditions, pH, etc.). An anti-connection sequence can be a reverse complementary sequence (or complementary sequence) of a connection sequence. When a connection sequence hybridizes with an anti-connection sequence, the base pairs formed can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more base pairs. The base pairs formed between a connection sequence and an anti-connection sequence can be continuous or non-continuous. In some embodiments, the first nucleic acid molecule comprises a ligation sequence, wherein the first nucleic acid molecule comprises a ligation sequence, wherein the second ... The length of the connecting sequence (or anti-connection sequence) can be less than or equal to about 300, 250, 200, 150, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 nucleotides. The connecting sequence (or anti-connection sequence) can be at the 5' end or 3' end of the nucleic acid molecule. The connecting sequence (or anti-connection sequence) can also be the internal sequence of the nucleic acid molecule. For example, the connecting sequence can be an internal connecting sequence, and can be exposed at the 5' end or 3' end by cutting the internal sequence of the nucleic acid molecule (for example, a sequence adjacent to the internal connecting sequence). The example of the internal connecting sequence is provided in Example 1, the interchain connecting sequence (ICC). In some cases, the connecting sequence and the anti-connection sequence are used to connect the nucleic acid molecule of the CDR3 or its part of the coding TCR chain to another nucleic acid molecule comprising a TCR V gene or its part. In some cases, linker sequences and anti-linker sequences are used to link a nucleic acid molecule comprising a TCR J region to another nucleic acid molecule comprising a TCR V gene or a portion thereof.In some cases, the linker and anti-linker sequences are used to link a nucleic acid molecule comprising a sequence encoding a CDR3 or a portion thereof and a J region of a TCR to another nucleic acid molecule comprising a TCR V gene or a portion thereof. In some cases, the linker and anti-linker sequences are used to link a nucleic acid molecule comprising a sequence encoding a CDR3 or a portion thereof, a J region and a TCR V gene or a portion thereof to another nucleic acid molecule encoding a constant domain of a TCR or a portion thereof.

[0102] The connecting sequence (or anti-connection sequence) can be a sequence encoding a portion of a TCR V gene (e.g., a portion of a TCR V gene adjacent to a sequence encoding CDR3 in a rearranged gene). And in such cases, the connecting sequence and one or more other connecting sequences in the connecting sequence library can encode the same amino acid sequence (e.g., a conserved portion of a TCR V gene adjacent to CDR3). When the connecting sequence encodes a conserved portion of a TCR V gene, the connecting sequence can be codon-diverse, so that the connecting sequence can be used to specifically connect a nucleic acid molecule to another nucleic acid molecule, thereby generating a nucleic acid molecule encoding a homologous pair of structures of a TCR. In some embodiments, the connection sequence (or anti-connection sequence) comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more nucleotides of the TCR V gene adjacent to the sequence encoding CDR3 in the rearranged gene. Due to the specificity of the connection sequence and the anti-connection sequence, a library of nucleic acid molecules with different sequences encoding different TCRs can be constructed in batch reactions (for example, in the same compartment). The connection sequence can indicate which TCR V gene the sequence encoding CDR3 should be connected to according to the reference sequence (for example, the native sequence of the TCR chain determined by sequencing). The linking sequence (or anti-linking sequence) can be any (e.g., predetermined) sequence that may not encode a portion of a TCR V gene. In such cases, the arbitrary sequence can be removed after the two nucleic acid fragments are linked together.

[0103] Figure 7Describe the example (thin arrow) of connecting CDR3-J polynucleotide to the specified V gene germline polynucleotide using any connecting sequence (701) and anti-connecting sequence (702). Each V gene germline polynucleotide here has a partial double-stranded structure. The top chain (the right side of its 3' end in this figure) has a single-stranded region at its 3' end. The connecting sequence and the anti-connecting sequence can be single-stranded and can hybridize with each other. The connecting sequence and the anti-connecting sequence are only used for the purpose of specific hybridization, and may be completely unrelated to TCR, so they are arbitrary. After hybridization between the connecting sequence and the anti-connecting sequence, the 3' end of the top chain of the V gene germline polynucleotide can be hybridized with CDR3-J polynucleotide and can be extended by DNA polymerase. The number of nucleotides on the 3' end of the top chain of the V gene germline polynucleotide hybridized with CDR3-J polynucleotide can be 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15, or up to 20.

[0104] The nucleic acid fragment used to construct the nucleic acid molecule encoding TCR or part thereof may include a self-cleaving peptide. The self-cleaving peptide may be a 2A peptide, an intein or a hedgehog peptide. Examples of 2A peptides include, but are not limited to, P2A (e.g., sequence: ATNFSLLKQAGDVEENPGP (SEQ ID NO: 284)), E2A (e.g., sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 285)), F2A (e.g., sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 286)) and T2A (e.g., sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 287)) peptides.

[0105] The nucleic acid fragment for constructing the nucleic acid molecule encoding TCR or its part may include a restriction enzyme recognition site. For example, the restriction enzyme recognition site may be a recognition site of an IIS type restriction enzyme. Examples of IIS type restriction enzymes that can be used for the present disclosure include but are not limited to EarI, MnlI, PleI, AlwI, BbsI, BbvI, BcoDI, BsaI, BseRI, BsmAI, BsmBI, BspMI, Esp3I, HgaI, SapI, SfaNI, BbvI, BsmFI, BsrDI, BtsI, FokI, BseRI, HphI, MlyI and MboII. In some cases, two or more different restriction enzymes can be used in the nucleic acid construction process. In some cases, a restriction enzyme (e.g., BbsI, BbvI, BcoDI, BsaI, BsmBI, FokI, etc.) that produces a 4-bp 5' overhang can be used. In some cases, restriction enzymes that produce blunt ends or 3' overhangs (eg, BseRI, BsrDI, BtsI, MlyI, etc.) can be used.

[0106] The nucleic acid fragment used to construct the nucleic acid molecule encoding TCR or its part can be cyclized.For example, the two ends of the nucleic acid fragment can be connected by connection to make the nucleic acid fragment cyclized.The connection can be a flat end connection.It can be connected after using 5' to 3' exonuclease (for example, Gibson Assembly), 3' to 5' exonuclease (for example, sequence and ligase independent cloning or SLIC) or USER enzyme mixture (for example, USER friendly DNA recombination or USERec) to produce sticky ends.Other examples of cyclization methods include but are not limited to circular polymerase extension cloning (CPEC) and seamless connection cloning extract (SLiCE) assembly.Or, the two ends can be connected by overlapping PCR.A variety of ligases can be used for connection, for example, including but not limited to, T4 DNA ligase, T4 RNA ligase, Escherichia coli DNA ligase.

[0107] The nucleic acid fragments of the nucleic acid molecules for constructing the encoding TCR chain or its part can be chemically synthesized. For example, the nucleic acid fragments can be pre-synthesized by chip-based synthesis. In some cases, the length of the synthesized nucleic acid fragments can be equal to or greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400 or more nucleotides. In some cases, the length of the synthesized nucleic acid fragments can be equal to or less than about 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 nucleotides.

[0108] The two nucleic acid sequences encoding the two peptide chains of the TCR can be constructed in several directions, such as head to head, head to tail, and tail to tail. As described herein, "head" refers to the "5' end" of the sense nucleic acid chain and "tail" refers to the "3' end" of the sense nucleic acid chain. In some cases, the direction is head to tail, which can control the order of the paired nucleic acid sequences encoding the TCR (e.g., TRA followed by TRB, or TRB followed by TRA).

[0109] Any nucleic acid molecule described herein can be a double-stranded nucleic acid molecule or a single-stranded nucleic acid molecule. In some cases, a nucleic acid molecule can include a double-stranded region and a single-stranded region. For example, a nucleic acid molecule with a connection sequence or an anti-connection sequence can be a double-stranded nucleic acid molecule with a connection sequence or an anti-connection sequence region as a single-stranded region (e.g., an overhang or a sticky end). The length of the overhang can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. The overhang can be located at the 5' end or 3' end of the nucleic acid molecule.

[0110] Any nucleic acid molecule described herein may comprise one or more modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl braid, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- Methoxyaminomethyl-2-thiouracil, β-D-mannosyl braided glycoside, 5'-methoxycarboxymethyl uracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyl adenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, braided glycoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, 2,6-diaminopurine, etc. In some cases, the nucleotide may contain modifications of its phosphate portion, including modifications to the triphosphate portion. Non-limiting examples of such modifications include larger length phosphate chains (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., α-thiotriphosphates and β-thiotriphosphates). Nucleic acid molecules can also be modified at the base moiety (e.g., at one or more atoms that are normally available for hydrogen bonding with complementary nucleotides and / or at one or more atoms that are normally unable to hydrogen bond with complementary nucleotides), the sugar moiety, or the phosphate backbone. Nucleic acid molecules can also contain amine-modified groups, such as aminoallyl 1-dUTP (aa-dUTP) and aminohexyl acrylamide-dCTP (aha-dCTP), to allow covalent attachment of amine-reactive moieties, such as N-hydroxysuccinimide ester (NHS). Substitution of standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure may provide higher density (in bits / m3), higher safety (resistance to accidental or purposeful synthesis of natural toxins), easier discrimination of photo-programmed polymerases, or lower secondary structure. Such substituted base pairs may be compatible with natural and mutant polymerases for de novo synthesis and / or amplification synthesis.

[0111] An exemplary workflow for constructing a nucleic acid molecule encoding a TCR is as follows Figure 1A-Figure 1CAs shown. Before constructing nucleic acid molecules encoding TCRs using the methods described herein, multiple homologous pairs of TCRs can be predetermined using various existing methods (e.g., single-cell barcoding and sequencing). Various sequencing methods can be used to determine the sequence of paired TCR chains, such as Sanger sequencing, high-throughput sequencing, synthetic sequencing, single-molecule sequencing, ligation sequencing, RNA-Seq (Illumina), next-generation sequencing, digital gene expression (Helicos), clonal single microarray (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, or massively parallel sequencing. The paired sequences from the sequencing library can be used as reference sequences for homologous pairs of TCR chains, so that which CDR3 is paired with which V gene can be known through the specific interaction between the connecting sequence and the anti-connection sequence. Multiple nucleic acid molecules encoding different TCRs can be constructed in batches using the methods described herein, but Figure 1A-Figure 1C The construction of one molecule is shown in as an example. A first nucleic acid molecule comprising a sequence encoding a first CDR3 (e.g., CDR3α) and a second CDR3 (e.g., CDR3β) can be contacted with a second nucleic acid molecule comprising a sequence derived from a first TCR V gene (e.g., TRAV). The connecting sequence (e.g., ConA#*) of the first nucleic acid molecule can hybridize with the anti-connection sequence (ConA#) of the second nucleic acid molecule to connect the two nucleic acid molecules. Extension and connection can be performed to produce a third nucleic acid molecule, which comprises a sequence derived from the first TCR V gene and a sequence encoding the first CDR3 and the second CDR3. Next, restriction enzymes (e.g., Figure 1AThe third nucleic acid molecule can be contacted with the fourth nucleic acid molecule comprising a sequence encoding the first constant region or constant domain (e.g., TRBC). The third nucleic acid molecule can then be connected to the fourth nucleic acid molecule by the overhang to produce the fifth nucleic acid molecule, which comprises a sequence derived from the first TCR V gene, a sequence encoding the first CDR3 and the second CDR3, and a sequence encoding the first constant region. The fifth nucleic acid molecule can be cyclized and cut with a restriction enzyme (e.g., TIISRE3) to expose an internal connection sequence (e.g., ICC). The fifth nucleic acid molecule can be contacted with the sixth nucleic acid molecule comprising a sequence derived from the second TCR V gene (e.g., TRBV). The fifth nucleic acid molecule can be connected to the sixth nucleic acid molecule by the interaction between the connecting sequence and the anti-connection sequence. The sixth nucleic acid molecule can be cut by a restriction enzyme (e.g., TIISRE2) to produce an overhang. The sixth nucleic acid molecule can be contacted with the seventh nucleic acid molecule comprising a sequence encoding the second constant region or constant domain (e.g., TRAC). The 6th nucleic acid molecule and the 7th nucleic acid molecule can be connected to form the 8th nucleic acid molecule comprising all regions of the coding pairing TCR chain. The 8th nucleic acid molecule can be further constructed into the expression vector for expressing the TCR chain in the host cell. It should be understood that the nucleic acid fragment comprising the sequence derived from the TCR V gene can be single-stranded, and in this case, the 3' end of the connection sequence of the nucleic acid fragment encoding CDR3 can be extended after hybridization with the anti-connection sequence.

[0112] The methods described herein can be used to generate libraries of single TCR chains, such as libraries of TCR alpha chains or TCR beta chains.

[0113] The method for producing a plurality of nucleic acid molecules as described herein may include providing a first plurality of nucleic acid molecules (or nucleic acid fragments). The nucleic acid molecules in the first plurality of nucleic acid molecules may include a sequence encoding the first CDR3 of the first T cell receptor (TCR) chain and the second CDR3 of the second TCR chain. The first CDR3 and the second CDR3 may be from the homology pairs of TCR chains. Then, a second plurality of nucleic acid molecules may be provided. The nucleic acid molecules in the second plurality of nucleic acid molecules may include a sequence derived from a TCR V gene. The nucleic acid molecules may not include a sequence encoding a constant domain. Then, the first plurality of nucleic acid molecules may be contacted with the second plurality of nucleic acid molecules. The nucleic acid molecules in the first plurality of nucleic acid molecules may be connected with the nucleic acid molecules in the second plurality of nucleic acid molecules to form a nucleic acid molecule comprising a sequence encoding the first CDR3 and the second CDR3 and a sequence derived from a TCR V gene. The sequence encoding the first CDR3 and the second CDR3 and the TCR V gene may be derived from the homology pairs of TCR chains.

[0114] The method for producing a plurality of nucleic acid molecules (each of which encodes a T cell receptor (TCR) chain or region thereof) may include contacting a first plurality of nucleic acid molecules with a second plurality of nucleic acid molecules to produce a third plurality of nucleic acid molecules comprising at least two different nucleic acid molecules. Each of the at least two different nucleic acid molecules may have different sequences encoding different TCR chains or regions thereof. At least two different nucleic acid molecules may be produced in the same compartment. In some cases, at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 15,000, 20,000, 100,000, 1,000,000, 10,000,000 or more different sequences encoding different TCRs can be produced in the same compartment.

[0115] The method for producing a variety of nucleic acid molecules as described herein may include providing a first variety of nucleic acid molecules. The nucleic acid molecules in the first variety of nucleic acid molecules may include the sequence of the first CDR3 of the first T cell receptor (TCR) chain and the second CDR3 of the second TCR chain. The first CDR3 and the second CDR3 can be from the homology of the TCR chain. Then, a second variety of nucleic acid molecules can be provided. The nucleic acid molecules in the second variety of nucleic acid molecules may include the sequence derived from the TCR V gene. Then, the first variety of nucleic acid molecules can be contacted with the second variety of nucleic acid molecules. The nucleic acid molecules in the first variety of nucleic acid molecules can be connected with the nucleic acid molecules in the second variety of nucleic acid molecules to form a linear nucleic acid molecule comprising the sequence encoding the first CDR3 and the second CDR3 and the sequence derived from the TCR V gene. The sequence encoding the first CDR3 and the second CDR3 and the TCR V gene can be derived from the homology of the TCR chain.

[0116] The method for producing a plurality of nucleic acid molecules may include providing a first plurality of nucleic acid molecules. The nucleic acid molecules in the first plurality of nucleic acid molecules may include a synthetic sequence of the first CDR3 of the first T cell receptor (TCR) chain and the second CDR3 of the second TCR chain (i), and (ii) a synthetic sequence of the third CDR3 of the third T cell receptor (TCR) chain and the fourth CDR3 of the fourth TCR chain. The first CDR3 and the second CDR3 may be from the first homology pair of the TCR chain, and the third CDR3 and the fourth CDR3 may be from the second homology pair of the TCR chain. Then, a second plurality of nucleic acid molecules may be provided. The nucleic acid molecules in the second plurality of nucleic acid molecules may include a sequence derived from a TCR V gene. Then, the first plurality of nucleic acid molecules may be contacted with the second plurality of nucleic acid molecules. The nucleic acid molecules in the first plurality of nucleic acid molecules may be connected with the nucleic acid molecules in the second plurality of nucleic acid molecules to form a nucleic acid molecule comprising a sequence encoding the first CDR3 and the second CDR3 and a sequence derived from a TCR V gene. The sequences encoding the first CDR3 and the second CDR3 and the TCR V gene may be derived from the homology pair of the TCR chain.

[0117] The method for producing the nucleic acid molecule of coding T cell receptor (TCR) chain or its part may include providing at least one nucleic acid molecule comprising the sequence of coding TCR chain CDR3.Then, a variety of nucleic acid molecules may be provided.Described multiple each kind of nucleic acid molecule may include the sequence derived from TCR V gene.Described multiple nucleic acid molecules may include at least two different sequences derived from at least two different TCR V genes.In some cases, multiple nucleic acid molecules may include at least 2,5,10,15,20,25,30,35,40 kinds or more different sequences derived from at least 2,5,10,15,20,25,30,35,40 kinds or more different TCR V genes.Then, at least one nucleic acid molecule comprising the sequence of coding TCR chain CDR3 may be contacted with multiple nucleic acid molecules (each kind of comprising the sequence derived from TCR V genes) in the same compartment. At least one nucleic acid molecule comprising a sequence encoding CDR3 of a TCR chain can be connected to a nucleic acid molecule from a plurality of nucleic acid molecules to produce a third nucleic acid molecule comprising a sequence encoding CDR3 and a sequence derived from one of at least two different TCR V genes, thereby producing a nucleic acid molecule encoding a TCR chain or a portion thereof.

[0118] Compositions described herein that can be used for methods described herein may include a first plurality of nucleic acid molecules. Each nucleic acid molecule in the first plurality of nucleic acid molecules may include a sequence encoding the first CDR3 of a first T cell receptor (TCR) chain and the second CDR3 of a second TCR chain. The first CDR3 and the second CDR3 may be from a homology pair of TCR chains. Compositions may also include a second plurality of nucleic acid molecules. Each nucleic acid molecule in the second plurality of nucleic acid molecules may include a sequence derived from a TCR V gene. Each nucleic acid molecule in the second plurality of nucleic acid molecules may not include a sequence encoding the first CDR3 and the second CDR3. In this composition, each nucleic acid molecule in (i) the first plurality of nucleic acid molecules may include a sequence encoding different first CDR3 and / or the second CDR3, and / or each nucleic acid molecule in (ii) the second plurality of nucleic acid molecules includes a sequence derived from different TCR V genes.

[0119] Compositions described herein that can be used for methods described herein can include a variety of nucleic acid molecules. Each of the multiple nucleic acid molecules can include a sequence derived from a T cell receptor (TCR) V gene. The multiple nucleic acid molecules can include a first nucleic acid molecule having a first connection sequence and a second nucleic acid molecule having a second connection sequence. The first connection sequence can be different from the second connection sequence.

[0120] The compositions described herein that can be used for the methods described herein can include a plurality of nucleic acid molecules. Each of the plurality of nucleic acid molecules can encode a CDR3 of a T cell receptor (TCR) chain. The first nucleic acid molecule in the plurality can include a first connection sequence, and the second nucleic acid molecule in the plurality can include a second connection sequence. The first connection sequence can be different from the second connection sequence.

[0121] The compositions described herein that can be used for the methods described herein may include a variety of nucleic acid molecules. Each of the multiple nucleic acid molecules may include a sequence of at least ten amino acids encoding a T cell receptor (TCR) chain. The first nucleic acid molecule in the multiple may include a first connecting sequence, and the second nucleic acid molecule in the multiple may include a second connecting sequence. The first connecting sequence may be different from the second connecting sequence. The first connecting sequence or the second connecting sequence may encode a portion of a TCR chain. The first connecting sequence or the second connecting sequence may be in frame with a sequence of at least ten amino acids encoding a TCR chain.

[0122] Compositions described herein that can be used for methods described herein may include a variety of nucleic acid molecules. Each nucleic acid molecule in the various nucleic acid molecules may include a sequence derived from a T cell receptor (TCR) V gene, and may not include a CDR3 sequence. The first nucleic acid molecule in the various may include a first anti-connection sequence, and the second nucleic acid molecule in the various may include a second anti-connection sequence. The first anti-connection sequence may be different from the second anti-connection sequence. The sequence of the TCR V gene derived from the first nucleic acid molecule and the second nucleic acid molecule may be derived from different TCR V genes. The composition may also include at least one nucleic acid molecule containing a sequence encoding the CDR3 of a TCR chain. The at least one nucleic acid molecule may also include a first connection sequence complementary to the first anti-connection sequence.

[0123] The present disclosure provides compositions and methods for assembling or synthesizing a TCR library comprising a variety of TCRs. In some cases, it may be useful to separate or purify a specific TCR sequence (e.g., target TCR) from a TCR library for further characterization or manipulation. For this reason, a barcode may be included in a nucleic acid molecule or fragment for constructing a sequence encoding TCR or a portion thereof. In some cases, a nucleic acid fragment comprising a sequence encoding CDR3 comprises a barcode. In some cases, a nucleic acid fragment comprising a sequence encoding the first CDR3 of the first TCR chain and the second CDR3 of the second TCR chain comprises a barcode. For example, a CDR3-J oligomer or a paired CDR3-J oligomer may include a barcode. A connection sequence (or in some cases, an anti-connection sequence) may include a barcode. The interchain connection sequence (or ICC) of a CDR3-J oligomer may include a barcode. A barcode may be a primer binding site, such as a TCR specific primer binding site or DOPBS.

[0124] For example, a library of oligomers encoding paired CDR3-J (e.g., Figure 1A) in a unique paired CDR3-J library can contain a unique barcode (or unique DOPBS). The sequence of DOPBS can be arbitrarily designed. The sequence of DOPBS can be designed to avoid common defects, such as unwanted secondary structures, restriction sites, similarities with other sequences in TCR genes, or similarities between primer binding sites. The length of the barcode (or DOPBS) can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides. DOPBS can be an additional sequence included in each sequence of the paired CDR3-J library. DOPBS can be a sequence already included in each sequence of the paired CDR3-J library. For example, the linker sequence or a portion thereof can be used as DOPBS. The sequences listed in Table 3 can be used as DOPBS. Figure 1C The product of step (9) can be used as a template in dial-out PCR using a forward primer corresponding to T2A-3 and a reverse primer corresponding to DOPBS associated with the target TCR. Figure 1C The final product may mainly contain the target TCR.

[0125] TCR expression

[0126] Using the method provided herein, the nucleic acid molecule library encoding each TCR or part thereof can be further delivered to the host cell for expression. The nucleic acid molecule constructed can be inserted into a vector so as to be expressed in a host cell. The nucleic acid molecule constructed can be delivered to a recipient cell as a linear or circular nucleic acid chain. In some cases, the nucleic acid constructed or the vector comprising the nucleic acid constructed can be delivered to the recipient cell by electroporation. In some cases, the nucleic acid constructed or the vector comprising the nucleic acid constructed can be delivered by a carrier (such as a cationic polymer).

[0127] The vector can be a plasmid, a transposon (e.g., Sleeping Beauty, Piggy Bac), an adenoviral vector, an AAV vector, a retroviral vector, or a lentiviral vector. Non-limiting examples of vectors include plasmids, shuttle vectors, phagemids, cosmids, virions, retroviral vectors, adenoviral vectors, or particles and / or vectors commonly used in gene therapy. Non-limiting examples of suitable plasmid vectors include pUC, pBR322, pET, pBluescript, and variants thereof. In addition, the vector may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selective marker sequences (e.g., antibiotic resistance genes), replication origins, and the like. The vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as a replication origin. The vector may also include one or more selectable marker genes and other genetic elements. The vector may be an expression vector comprising a nucleic acid sequence encoding a TCR or a portion thereof constructed according to the present disclosure that is operably linked to a sequence that allows TCR expression. Other examples of vectors include, but are not limited to, viral and non-viral vectors, such as retroviral vectors (including lentiviral vectors), adenoviral vectors (including replicating, replication-defective and enterotypes thereof), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Moloney murine leukemia vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors and non-viral plasmids. Baculovirus vectors may be suitable for expression in insect cells. Non-viral vectors may be formulated as nanoparticles, cationic lipids, cationic polymers, metal nanopolymers, nanorods, liposomes, micelles, microvesicles, cell penetrating peptides or lipid spheres.

[0128] In some embodiments, the vector is a self-amplifying RNA replicon, also referred to as self-replicating (m) RNA, self-replicating (m) RNA, self-amplifying (m) RNA or RNA replicon. The self-amplifying RNA replicon is an RNA that can self-replicate. In some embodiments, the self-amplifying RNA replicon can self-replicate in cells. In some embodiments, the self-amplifying RNA replicon encodes RNA polymerase and target molecule. The RNA polymerase can be an RNA-dependent RNA polymerase (RDRP or RdRp). The self-amplifying RNA replicon can also encode a protease or an RNA capping enzyme. In some embodiments, the self-amplifying RNA replicon vector belongs to or is derived from a virus of the Togaviridae family known as alphaviruses, which may include Eastern equine encephalitis virus (EEE), Venezuelan equine encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixunavirus, Western equine encephalitis virus (WEE), Sindbis virus, South African arbovirus 86, Semliki Forest virus, Middelburg virus, Chikungunya virus, Onyong-nyong virus, Ross River virus, Bama Forest virus, Gaita virus, Lushan virus, Bebaru virus, Mayaro virus, Una virus, Ora virus, Vodaro River virus, Babanki virus, Kyzylagach virus, Highland J virus, Fort Morgan virus, Ndumau virus, Buggy Creek virus, and any other virus classified as an alphavirus by the International Committee on Taxonomy of Viruses (ICTV). In some embodiments, the self-amplifying RNA replicon is or contains a portion of an attenuated form of an alphavirus, such as the VEE TC-83 vaccine strain. In some embodiments, the self-amplifying RNA replicon vector has been engineered or selected in vitro, in vivo, ex vivo or in silico for a specific function (e.g., extended or increased expression of a dichotomous immune receptor) in a host cell, a target cell, or an organism. For example, a host cell population carrying different variants of a self-amplifying RNA replicon can be selected based on the expression level of one or more target molecules (encoded in the self-amplifying RNA replicon or the host genome) at different time points. In some embodiments, the selected or engineered self-amplifying RNA replicon has been modified to reduce type I interferon responses, innate antiviral responses, or adaptive immune responses from a host cell or an organism, which results in the protein expression of the RNA replicon continuing longer or expressing at a higher level in a host cell, a target cell, or an organism.In some embodiments, this optimized self-amplifying RNA replicon sequence is obtained from a single cell or cell population having a desired phenotypic trait (e.g., higher or more persistent expression of a target molecule compared to a wild-type strain or vaccine strain, or reduced innate antiviral immune response to a vector). In some embodiments, cells carrying a desired or selected self-amplifying RNA replicon sequence are obtained from subjects (e.g., humans or animals) (e.g., elite responders or subjects of complete remission) having beneficial response characteristics after treatment with a therapeutic agent comprising a self-amplifying RNA replicon. In some embodiments, the self-amplifying RNA replicon may contain one or more subgenomic sequences to produce one or more subgenomic polynucleotides. In some embodiments, the subgenomic polynucleotides serve as functional mRNA molecules for translation by cellular translation mechanisms. Subgenomic polynucleotides can be produced by the function of a determined sequence element (e.g., a subgenomic promoter or SGP) on a self-amplifying RNA replicon, and the determined sequence element directs a polymerase to produce subgenomic polynucleotides from a subgenomic sequence. In some embodiments, SGP is recognized by RNA-dependent RNA polymerase (RDRP or RdRp). In some embodiments, multiple SGP sequences are present on a single self-amplification RNA replicon, and can be located upstream of the subgenomic sequence encoding a dichotomous immune receptor, a component of a dichotomous immune receptor, or other agents. In some embodiments, the nucleotide length or composition of the SGP sequence can be modified to change the expression characteristics of the subgenomic polynucleotide. In some embodiments, non-identical SGP sequences are located on the self-amplification RNA replicon, so that the ratio of the corresponding subgenomic polynucleotide is different from the situation that the SGP sequence is identical. In some embodiments, non-identical SGP sequences guide the generation of TCR and other agents (such as cytokines) so that they are produced relative to each other at a certain ratio, thereby causing TCR expression to increase, target cell amplification to increase or faster and to target cells or hosts without cytotoxic effects, or weakening the innate or adaptive immune response for RNA replicons. In some embodiments, the position of subgenomic sequences and SGP sequences relative to each other and the genome sequence itself can be used to change the ratio of subgenomic polynucleotides relative to each other. In some embodiments, the SGP and subgenomic sequences encoding TCR can be located downstream of the SGP and subgenomic regions encoding other agents, so that the expression of TCR is greatly increased relative to other agents. In some embodiments, the RNA replicon or SGP has been selected or engineered to express an optimal amount of a cytokine such that the cytokine promotes the expansion of T cells or enhances the therapeutic effect of the TCR but does not cause severe side effects such as cytokine release syndrome, cytokine storm, or neurotoxicity.

[0129] The expression of two chains can be driven by two promoters or one promoter. In some cases, two promoters are used. In some cases, two promoters together with the protein coding sequences of their two chains can be arranged in a head-to-head, head-to-tail or tail-to-tail direction. In some cases, a promoter is used. Two protein coding sequences can be connected in frame so that a promoter can be used to express two chains. And in such cases, two protein coding sequences can be arranged in a head-to-tail direction and can be connected to a ribosome binding site (such as an internal ribosome binding site or IRES), a protease cleavage site or a self-processing cleavage site (such as a sequence encoding a 2A peptide) to promote bicistronic expression. In some cases, two chains can be connected with a peptide linker so that two chains can be expressed as single-chain polypeptides. Each chain expressed can contain a complete variable domain sequence comprising a rearranged V (D) J gene. Each chain expressed can contain a complete variable domain sequence, including CDR1, CDR2 and CDR3. Each expressed chain may contain a complete variable domain sequence, including FR1, CDR1, FR2, CDR2, FR3 and CDR3. In some cases, each expressed chain may also contain a constant domain sequence.

[0130] To generate an expression vector, additional sequences can be added to the constructed nucleic acid molecule. These additional sequences include a vector backbone (e.g., elements required for the vector to replicate in a target cell or temporary host (such as E. coli)), a promoter, an IRES, a sequence encoding a self-cleaving peptide, a terminator, an auxiliary gene (such as a payload), and a partial sequence of an immune receptor polynucleotide (such as a portion of a sequence encoding a constant domain).

[0131] Protease cleavage sites include, but are not limited to, enterokinase cleavage site: (Asp)4Lys (SEQ ID NO: 288); factor Xa cleavage site: Ile-Glu-Gly-Arg (SEQ ID NO: 289); thrombin cleavage site, such as Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 290); renin cleavage site, such as His-Pro-Phe-His-Leu-Val-Ile-His (SEQ ID NO: 291); NO:291); collagenase cleavage site, such as X-Gly-Pro (wherein X is any amino acid); trypsin cleavage site, such as Arg-Lys; viral protease cleavage site, such as viral 2A or 3C protease cleavage site, including but not limited to protease 2A cleavage site from picornavirus, hepatitis A virus 3C cleavage site, human rhinovirus 2A protease cleavage site, picornavirus 3 protease cleavage site; and caspase protease cleavage site, such as DEVD (SEQ ID NO:292) recognized and cleaved by activated caspase-3, wherein cleavage occurs after the second aspartic acid residue. In some embodiments, the present disclosure provides an expression vector comprising a protease cleavage site, wherein the protease cleavage site comprises a cellular protease cleavage site or a viral protease cleavage site. In some embodiments, the first protein cleavage site comprises a site recognized by furin; VP4 of IPNV; tobacco etch virus (TEV) protease; 3C protease of rhinovirus; PC5 / 6 protease; PACE protease, LPC / PC7 protease; enterokinase; factor Xa protease; thrombin; gene enzyme I; MMP protease; nuclear inclusion protein a (N1a) of turnip mosaic potyvirus; NS2B / NS3 of Dengue type 4 flaviviruses, NS3 protease of yellow fever virus; ORF V of cauliflower mosaic virus; KEX2 protease; CB2; or 2A. In some embodiments, the protein cleavage site is a virus-internal cleavable signal peptide cleavage site. In some embodiments, the virus-internal cleavable signal peptide cleavage site comprises a site from influenza C virus, hepatitis C virus, hantavirus, flavivirus, or rubella virus.

[0132] Suitable IRES elements included in the vectors of the present disclosure may include RNA sequences capable of engaging eukaryotic ribosomes. In some embodiments, the IRES elements of the present disclosure are at least about 250 base pairs, at least about 350 base pairs, or at least about 500 base pairs. The IRES elements of the present disclosure may be derived from the DNA of an organism, including but not limited to viruses, mammals, and fruit flies. In some cases, the viral DNA from which the IRES elements are derived includes but is not limited to picornavirus complementary DNA (cDNA), encephalomyocarditis virus (EMCV) cDNA, and poliovirus cDNA. Examples of mammalian DNA from which IRES elements are derived include but are not limited to DNA encoding immunoglobulin heavy chain binding protein (BiP) and DNA encoding basic fibroblast growth factor (bFGF). Examples of fruit fly DNA from which IRES elements are derived include but are not limited to the Antennapedia gene from Drosophila melanogaster. Other examples of poliovirus IRES elements include, for example, poliovirus IRES, encephalomyocarditis virus IRES, or hepatitis A virus IRES. Examples of Flavivirus IRES elements include a Hepatitis C virus IRES, a GB virus B IRES, or a Pestivirus IRES, including but not limited to a Bovine Viral Diarrhea Virus IRES or a Classical Swine Fever Virus IRES.

[0133] Examples of self-processing cleavage sites include, but are not limited to, intein sequences; modified inteins; hedgehog sequences; other porcine sequences; 2A sequences, such as a 2A sequence derived from foot-and-mouth disease virus (FMDV); and variants thereof.

[0134] The vector for recombinant immunoglobulin or other protein expression can include any number of promoters, wherein the promoter is constitutive, regulated or inducible, cell type specific, tissue specific or species specific. Other examples include tetracycline responsive promoters. The vector can be a replicon of a host cell suitable for expressing a recombinantly constructed gene, and it can include a replicon that is functional in a bacterial cell (e.g., Escherichia coli). The promoter can be constitutive or inducible, wherein induction is associated with, for example, a specific cell type or a specific maturity level. Alternatively, many viral promoters may be suitable. Examples of promoters include β-actin promoters, SV40 early and late promoters, immunoglobulin promoters, human cytomegalovirus promoters, retroviral promoters, elongation factor 1A (EF-1A) promoters, phosphoglycerate kinase (PGK) promoters, and Fried (Friend) spleen focus formation virus promoters. The promoter may or may not be associated with an enhancer, wherein the enhancer may be naturally associated with a specific promoter or associated with a different promoter.

[0135] application

[0136] Compositions and methods described herein may have various applications. Exemplary applications may be to reconstruct the sequence of the TCR encoding natural pairing by sequencing data (e.g., single cell sequencing data). In some applications, people may wish to reconstruct the sequence of the TCR encoding natural pairing identified from tumor infiltrating T cells. In these applications, fresh tissue samples (e.g., fresh solid tumor samples) from subjects can be used for single cell sequencing to obtain sequence information of two TCR chains of natural paired TCR. However, when the tissue sample containing tumor infiltrating cells (e.g., solid material sample that is not a body fluid sample) is a frozen sample or a fixed sample (e.g., FFPE sample), it may be challenging to separate cells to obtain a single cell suspension. In these cases, blood samples containing peripheral T cells from the same subject can be used for single cell sequencing to identify the sequence of the TCR of natural pairing. Because the blood sample may contain tumor infiltrating T cells released from the tissue sample into the bloodstream, the sequence obtained from the blood sample may contain sequences from these tumor infiltrating T cells. Then, tissue samples from the same subject can be used for batch sequencing. Although the batch sequencing of tissue samples may not provide the paired sequence of naturally paired TCRs, it can provide the CDR3 sequence of a single TCR chain. Then, the CDR3 sequence obtained in the batch sequencing of tissue samples (referred to herein as "tissue CDR3 sequence") can be used to compare with the paired sequence obtained in the single cell sequencing of blood samples. If the CDR3 sequence of the paired sequence matches the tissue CDR3 sequence, the paired sequence can be identified and used for any downstream application.

[0137] Single-cell sequencing refers to obtaining sequence information from a single cell. In single-cell sequencing, a cell population can be made into a single-cell suspension and separated into separate partitions. In each partition, the sequence released from a single cell can be barcoded and then sequenced. Various single-cell sequencing methods can be used for TCR reconstruction (see De Simone M, Rossetti G and Pagani M (2018) Single Cell T Cell Receptor Sequencing: Techniques and Future Challenges. Front. Immunol. 9: 1638). Batch sequencing refers to obtaining sequence information from a cell population. In batch sequencing, nucleic acid molecules can be separated from a mixture of cells and sequenced together.

[0138] Fig. 9AAn exemplary workflow for identifying tumor-infiltrating TCRs in a tumor sample using a blood sample is shown. First, a blood sample can be drawn from a patient. Next, a PBMC sample containing peripheral blood mononuclear cells can be separated from the blood sample. For example, these cells can be extracted from whole blood using ficoll (a hydrophilic polysaccharide that separates the layers of blood) and gradient centrifugation. Next, T cells can be isolated from the PBMC sample. The mononuclear cells can be removed by lysing the red blood cells, for example, by PERCOLL TM Gradient centrifugation or by countercurrent centrifugal elutriation, T cells are separated from PBMC. Optionally, T cell subsets can be further enriched by sorting based on markers. The marker can be a cell surface marker. Examples of cell surface markers include but are not limited to CD39, CD69, CD103, CD25, PD-1, TIM-3, OX-40, 4-1BB, CD137, CD3, CD28, CD4, CD8, CD45RA, CD45RO, GITR and FoxP3. The marker can be a cytokine. Examples of cytokine markers include but are not limited to IFN-γ, TNF-α, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B and perforin. Single cell sequencing can then be performed on T cells or T cell subsets to obtain the paired sequence of the naturally paired TCR (e.g., Fig. 9A The tumor sample can also be obtained from the same patient. The tumor sample can be a fixed or frozen sample. For example, the tumor sample can be fixed by a fixative such as formaldehyde. The tumor sample can be a formalin-fixed paraffin-embedded (FFPE) tissue sample. The tumor sample can then be batch sequenced to obtain the CDR3 sequence of the TCR chain. The CDR3 sequence obtained from the tumor sample can then be used to compare with the CDR3 sequence of the paired sequence to identify tumor-infiltrating TCRs. Tumor-infiltrating TCRs can be expressed in normal T cells or cell lines, which are expressed in Fig. 9A Shown as a “virtual TIL”.

[0139] Fig. 9BThe exemplary application of virtual TIL is shown.Virtual TIL may include a reporter system, which can be used for the T cell selection based on the reporter of target reactive TCR.For example, virtual TIL can be a reporter cell comprising a reporter gene, and when the TCR of the cell is combined with the target antigen, the reporter gene is adjusted to send a signal.These virtual TILs can be activated by contacting with antigen presenting cells (APC) or artificial APC loaded with antigen.Then, the target reactive T cells can be selected based on the signal or other selection mechanisms (for example, cell surface markers or cytokine markers) generated by the reporter system, for example, by FACS.The selection can be based on the cell surface marker expression on the virtual TIL after the antigen combined with the cell contact MHC.Cell surface markers can be CD25, CD69, CD39, CD103, CD137 and other T cell activation markers or any combination thereof.The selection can be based on calcium influx.The selection can also be based on reporter gene expression.The reporter gene can be a fluorescent protein (such as GFP and mCherry).The reporter gene can be under the control of a transcription factor regulated by TCR signal transduction. Examples of these transcription factors include, but are not limited to, AP-1, NFAT, NF-κ-B, Runx1, Runx3, etc. The selection can be based on cytokines released from activated virtual TILs using methods such as ICS and cytokine capture assays. Fig. 9C Another application of virtual TIL is shown. After identifying the target reactive TCR, the target reactive TCR can be delivered and expressed in host cells such as autologous T cells (T cells isolated from the same patient from which tissue samples and blood samples are obtained). The target reactive TCR can be delivered and expressed in allogeneic T cells. The T cells expressing the target reactive TCR can then be administered to the same patient to treat a disease, such as cancer.

[0140] The method for identifying the sequence of a naturally paired T cell receptor (TCR) in a tissue sample (e.g., a solid sample) from a subject can include identifying one or more paired sequences of one or more naturally paired TCRs in a sample containing multiple peripheral T cells obtained from a subject. Each of the one or more paired sequences can include a CDR3 sequence. Then, the tissue CDR3 sequence of the TCR chain of the TCR in the tissue sample can be identified, and other TCR chains with its natural pairing may be unknown. The tissue CDR3 sequence can match the CDR3 sequence of at least one paired sequence of one or more paired sequences of the TCR of one or more naturally paired TCRs, thereby identifying the at least one paired sequence as the sequence of the TCR naturally paired in the tissue sample. Also provided herein is a method for identifying a target reactive T cell receptor (TCR). The method may include providing a cell comprising a TCR identified using the method described herein. Then, the cell may contact a target antigen presented by an antigen presenting cell (APC). The cell can be combined with the target antigen presented by the APC by the TCR, thereby identifying the TCR as a target reactive TCR.

[0141] APC described herein can be a professional APC, such as a dendritic cell, a macrophage or a B cell. APC can be a monocyte or a monocyte-derived dendritic cell. aAPC can express ligands and co-stimulatory molecules of the T cell receptor, and can activate and amplify T cells for transfer, while improving their efficacy and function in some cases. aAPC can be engineered to express any gene for T cell activation. aAPC can be engineered to express any gene for T cell amplification. aAPC can be a bead, a cell, a protein, an antibody, a cytokine or any combination. aAPC can transmit a signal to a cell population that can undergo genome transplantation. For example, aAPC can transmit signal 1, signal 2, signal 3 or any combination. Signal 1 can be an antigen recognition signal. For example, signal 1 can be the connection of a TCR to a peptide-MHC complex or the combination of an agonist antibody to CD3, which can lead to the activation of a CD3 signal-transduction complex. Signal 2 can be a co-stimulatory signal. For example, the costimulatory signal can be anti-CD28, inducible costimulator (ICOS), CD27 and 4-1BB (CD137) that bind to ICOS-L, CD70 and 4-1BBL, respectively. Signal 3 can be a cytokine signal. The cytokine can be any cytokine. The cytokine can be IL-2, IL-7, IL-12, IL-15, IL-21 or any combination thereof.

[0142] In some cases, aAPC can be used to activate and / or expand cell populations. In some cases, artificial allotype specificity may not be induced. In some cases, aAPC may not express HLA. aAPC may be genetically modified to stably express genes that can be used for activation and / or stimulation. In some cases, K562 cells can be used for activation. K562 cells can also be used for expansion. K562 cells can be human erythroleukemia cell lines. K562 cells can be engineered to express target genes. K562 cells may not endogenously express HLA class I, class II or CD1d molecules, but may express ICAM-1 (CD54) and LFA-3 (CD58). K562 can be engineered to transmit signals 1 to T cells. For example, K562 cells can be engineered to express HLA class I. In some cases, K562 cells can be engineered to express additional molecules such as B7, CD80, CD83, CD86, CD32, CD64, 4-1BBL, anti-CD3, anti-CD3 mAb, anti-CD28, anti-CD28 mAb, CD1d, anti-CD2, membrane-bound IL-15, membrane-bound IL-17, membrane-bound IL-21, membrane-bound IL-2, truncated CD19, or any combination. In some cases, the engineered K562 cells may express anti-CD3 mAb, clone OKT3 in membrane form, in addition to CD80 and CD83. In some cases, the engineered K562 cells may express anti-CD3 mAb, clone OKT3, anti-CD28 mAb in membrane form, in addition to CD80 and CD83.

[0143] Reagent test kit

[0144] Compositions as described herein can be provided in a test kit.For example, a test kit may include a container with a nucleic acid molecule library that can be used to construct a variety of polynucleotide molecules, and each polynucleotide encodes a TCR chain or a homology pair of a TCR chain or a portion thereof. In some cases, each nucleic acid molecule of a nucleic acid molecule library encodes the CDR3 of a TCR chain. In some cases, each nucleic acid molecule of a nucleic acid molecule library encodes the first CDR3 and the second CDR3 of the homology pair of a TCR chain. In some cases, each nucleic acid molecule of a nucleic acid molecule library comprises a sequence derived from a TCR V gene. In some cases, each nucleic acid molecule of a nucleic acid molecule library comprises a connection sequence as described herein. Connection sequences may have sequences different from other connection sequences in the same nucleic acid molecule library. The test kit may include one or more containers, and each container contains a nucleic acid molecule library. The nucleic acid molecules provided in the test kit may be in liquid form or in a dried form (e.g., lyophilized form).

[0145] The kit may also contain instructional materials for directing the user to use the nucleic acid molecule library to construct a plurality of polynucleotide molecules encoding TCRs.

[0146] The kit may also include at least one reagent (eg, buffer, enzyme, additive, etc.) that can be used in a reaction to construct a nucleic acid molecule.

[0147] Example

[0148] Example 1. Conversion of a CDR3-J oligonucleotide library into a full-length, expressible TCR library

[0149] The present embodiment uses 3 types of IIS restriction enzymes to produce sticky ends. Such enzymes are commercially available. In the present embodiment, two enzymes (for example, BbsI, BbvI, BcoDI, BsaI, BsmBI, FokI, etc.) that produce 4-bp 5' overhangs and a restriction enzyme (for example, BseRI, BsrDI, BtsI, MlyI, etc.) that produces flat ends or 3' overhangs are used. The best enzyme group used can depend on actual factors (for example, local availability, cutting efficiency, star activity) and can be easily selected by experiment. Here, the first two restriction enzymes are called TIISRE1, TIISRE2, and the last restriction enzyme is called TIISRE3.

[0150] In the present embodiment, the paired CDR3-J oligonucleotides are synthesized in a "head to tail" direction relative to the coding sequences of α and βCDR3-J. In other words, αCDR-3J and βCDR-3J are synthesized in the same 5' to 3' direction. The full-length, expressible TCR polynucleotides produced are also head to tail directions. The paired CDR3-J oligonucleotides can be synthesized in other directions, such as head to head and tail to tail. The methods described herein can be combined with the methods described in U.S. Provisional Patent Application Nos. 62 / 718,227, 62 / 725,842, 62 / 732,898, 62 / 818,355 and 62 / 823,831 (each of these applications is incorporated herein by reference in its entirety) to design paired CDR-3J oligonucleotides and obtain full-length, expressible TCR polynucleotides in other directions.

[0151] like Figure 1A-Figure 1C As shown, the paired CDR3-J oligomer contains the reverse complementary sequences of TRBJ, CDR3β, TRAJ and CDR3α in 5' to 3' order, as well as other intervening domains described below. In this article, the symbol "*" indicates complementarity. For example, if P refers to a polynucleotide sequence, then P* refers to the reverse complementary sequence of P. In addition, the letter X is used to represent A or B when appropriate. For example, TRXV can be used to collectively refer to TRAV and TRBV. For clarity, in this embodiment and Figure 1A-Figure 1C In the present invention, the TRAJ domain and the TRBJ domain refer to polynucleotide sequences encoding parts of the TRAJ region and the TRBJ region, respectively, which are not included in the CDR3.

[0152] BCC Representatives "β Constant linking sequence", whose function is to connect with TRBC sequence. ConB# is a specific TR B Concatenated sequences of the V sequences, where the symbol # represents the numeric ID of the TRBV gene. Similarly, ConA# is a specific TR A Linking sequence of V sequences. ICC stands for "interchain linker sequence" and will be used to link TRBV to ConB#, and TRAC to TRAJ.

[0153] The ConB# and ConA# domains can be codon diversified (see Example 2) so that ConX# from different TRBV genes are sufficiently different at the nucleotide level that ConX# and ConX#* can hybridize with high yield only when the numerical IDs of ConX# and ConX#* are identical.

[0154] A library of 48 partially double-stranded TRAV#_GL polynucleotides (one annotated as functional for each TRAV gene in IMGT) can be prepared using conventional methods. All TRAV#_GL polynucleotides can be mixed to produce a TRAV#_GL library. GL stands for germline. The top strand of each TRAV#_GL polynucleotide contains (1) a P2A-3 domain, which encodes the 3' portion of the self-cleaving P2A peptide, (2) a TRAV#_GL5 domain, which encodes the 5' portion of the germline sequence of TRAV#, including, in this order, L, FR1, CDR1, FR2, CDR2, and a portion of FR3 upstream of ConA#, and (3) ConA#, which encodes the final fragment of FR3 and is codon-diverse. The bottom strand of each TRAV#_GL polynucleotide contains TRAV#_GL5* and P2A-3*. Thus, the TRAV#_GL polynucleotide has a 3' overhang of the sequence ConA#. Libraries and libraries of 48 TRBV#_GL polynucleotides can be prepared similarly. The P2A-3 domain in TRAV#_GL can be replaced by T2A-3 in TRBV#_GL. T2A is another self-cleaving peptide.

[0155] Libraries of 1,000 to 500,000 paired CDR3-J oligonucleotides can be prepared by chip-based synthesis.

[0156] In step (1), the TRAV#_GL library can be mixed with the paired CDR3-J library at a temperature that allows specific hybridization between ConA# and ConA#*. Then, in step (2), a DNA polymerase can be used to extend the top strand of TRAV#_GL, and a ligase can be used to connect the paired CDR3-J oligomers to the bottom strand of TRAV#_GL.

[0157] The BCC contains the recognition site for TIISRE1. In step (3), TIISRE1 can be used to cut at the BCC, leaving a 4-base 5' overhang on the bottom strand. In this embodiment, the 4 bases are antisense to the first 4 bases of TRBC1. In step (4), this cleavage product can be ligated to a pre-prepared TRBC_P2A-5_SE containing the complete TRBC1 sequence and the P2A-5 domain, and having a 4-base 5' overhang at the beginning of the TRBC1 sequence. The P2A-5 domain is the 5' end portion of the P2A coding sequence. SE represents a sticky end. This ligation product can be PCR amplified in step (5).

[0158] In step (6), the amplified product can be circularized by ligation between P2A-5 and P2A-3 using the methods described in U.S. Provisional Patent Application Nos. 62 / 718,227, 62 / 725,842, 62 / 732,898, 62 / 818,355, and 62 / 823,831. After ligation, P2A-5 and P2A-3 form P2A. In this embodiment, ICC contains a recognition site for TIISRE3, which can be used in step (7) to immediately cut the 3' of ConB#* on the bottom strand. The cleavage site on the top strand is less important. In step (8), the cleavage product can be heated to separate the top and bottom strands. A primer containing the first approximately 20 bases of TRBC1 can be used to extend on the bottom strand, leaving a single-stranded region at the 3' end of the bottom strand. At the tip of the 3' end of this strand is the ConB#* domain. In step (9), the TRBV#_GL library can be added so that the ConB# on the top strand of TRBV#_GL can hybridize with the corresponding ConB#*. DNA polymerase and ligase can be added to convert the hybridization product into a completely double-stranded DNA.

[0159] The remaining portion of ICC also contains a recognition site for TIISRE2, which can be used to cut ICC in step (10), leaving a 4-base 5' overhang, which is the antisense sequence of the first 4 bases of TRAC. In step (11), the pre-prepared TRAC_SE can be ligated to the 5' overhang to form a complete TRAC sequence, similar to step (4) above.

[0160] The final product can be ligated into a lentiviral backbone or appropriate “homologous sequence” for CRISPR / TALEN / ZFN-based knock-in.

[0161] Example 2. Testing codon diversification using human TRAV and TRBV sequences

[0162] In the present embodiment, a thermodynamically based algorithm is provided to design codon-diversified ConA# and ConB# sequences. The algorithm is written in MATLAB language. Some variables and custom functions used in the algorithm will be described in the "Notes" section below, and the rest are described in the comments of the code or are self-explanatory to those skilled in the art. Some custom functions rely on thermodynamically based simulations of DNA hybridization using publicly available thermodynamic parameters (e.g., ΔH and ΔS for base pair stacking) and models (e.g., ΔS as a function of loop size). These parameters and models have been widely published by John SantaLucia Jr. Technicians can easily write these functions from scratch or with the aid of publicly available software packages (such as NUPACK). The algorithm contains two stages: initial design and codon diversification, which are described in script 1 and script 2, respectively. In the initial design, ConA# and ConB# sequences are designed according to the original TRAV or TRBV sequences. The hybridization yield of each ConX# and each ConX#* is then calculated as a baseline ( Figure 2A and Figure 2B ). Figure 2A Shown are the hybridization yields of junction sequences designed based on the original TRAV sequence without codon diversification (ConA# vs. ConA#*). Figure 2B The hybridization yields of the junction sequences designed based on the original TRBV sequence without codon diversification (ConB# vs. ConB#*) are shown. During the codon diversification process, the codon selection of the last approximately 60 bases of some TRXV#_GLs was randomized, and ConX# sequences that allowed specific hybridization were selected. Next, the hybridization yields of each ConX# vs. each ConX#* of the sequence group using codon diversification were calculated to observe whether the codon diversification was successful ( Figure 3A and Figure 3B ). Figure 3A Hybridization yields of codon-diversified junction sequences (ConA# vs. ConA#*) are shown. Figure 3B Hybridization yields of codon-diversified junction sequences (ConB# vs. ConB#*) are shown.

[0163] Script 1: Initial design.

[0164]

[0165]

[0166]

[0167] Notes for Script 1:

[0168] The files "hsTRAV_UTR200-L-V_Sorted_FOnly.txt" and "hsTRBV_UTR 200-L-V_Sorted_FOnly.txt" are TSV files that record the sequences of all TCR V genes annotated as "functional" in the IMGT database. Each file has 4 columns, the first column is the name of the V gene, the fourth column is the V gene cDNA sequencing sequence starting from about 200nt upstream of the start codon (L-PART1), and the second column is the position of the first nucleotide of the start codon. The third column is the position of the first nucleotide of the V gene (for example, after L-PART2).

[0169] The function "fun_Design1on1" returns a ConA or ConB sequence using two inputs: (1) a variable cSA60 that records the last 60 bases of TRXV#_GL, and (2) parameters for thermodynamic modeling stored in a variable struPara. Briefly, the function finds the shortest contiguous subsequence of cSA60 (denoted as ConX) that ends at the 3' end of cSA60 that satisfies the following statement: When 5 nM of a first DNA oligonucleotide having the sequence ConX and 0.05 nM of a second DNA oligonucleotide having the sequence ConX* are mixed, more than 97% of the second oligonucleotide is expected to bind to the first oligonucleotide at the temperature, sodium ion concentration, and magnesium ion concentration defined by struPara.fHybTemp, struPara.fConcNa, and struPara.fConcMg, respectively. The output of this function (ra1on1ofThisGene) is a cell array with two cells, the first cell, ra1on1ofThisGene{1} is a 1x2 vector, where ra1on1ofThisGene{1}(1) is an output not used in this embodiment, and ra1on1ofThisGene{1}(2) is the position of the first base of ConX on cSA60. ra1on1ofThisGene{2} is a 1x4 cell array, where ra1on1ofThisGene{2}{1} and ra1on1ofThisGene{2}{2} are not used in this embodiment, ra1on1ofThisGene{2}{3} is the sequence of ConX, and ra1on1ofThisGene{2}{4} is the sequence of ConX*. One skilled in the art can write this function as described above.

[0170] When 5 nM (reported by fConcQB) of the second nucleotide and 0.05 nM (reported by fConcQB / 100) of the first nucleotide were mixed at 60 °C (reported by fHybTemp) and in 125 mM Na+ (recorded by fConcNa) ​​and 5mMMg ++ The function "NP_GetBoundFrac" returns the fraction of a first DNA oligonucleotide having sequence ConX* that is bound to a second DNA oligonucleotide having sequence ConX when equilibrium is reached in the presence of (recorded by fConcMg).

[0171] The image generated by this script shows a grayscale heat map showing how to predict the fraction of ConX#* that binds to ConX# when 0.05 nM ConX#* is mixed with 5 nM ConX# under the above conditions. Figure 2A and Figure 2B As shown, especially for TRBV ( Figure 2B ), there are a lot of cross-links (e.g., misconnections).

[0172] Script 2. Codon diversification

[0173]

[0174]

[0175]

[0176]

[0177] Notes for Script 2:

[0178] Using the codon table information provided by the input raCodonTable and the minimum allowed codon frequency provided by the input fCodonFreqThreshold, the function "fun_aa2nt" returns a polynucleotide sequence that encodes the same polypeptide as the input sequence cAAInFrame.

[0179] The image generated by this script shows a grayscale heat map of the fraction of ConX#* predicted to bind to ConX# when 0.05 nM ConX#* is mixed with 5 nM ConX# under the above conditions after codon diversification. Figure 3A and Figure 3B As shown, only specific hybridization is predicted to occur in large quantities. Thus, this example demonstrates that the codon diversification approach is feasible and shows how to obtain codon-diversified ConA and ConB sequences.

[0180] Example 3. Connecting sequences derived from mouse TRAV and TRBV genes

[0181] This example provides a codon-diverse junction sequence derived from mouse TRAV and TRBV genes. Similar to the above example, ConA is a specific TR AV sequence connecting sequence, ConB is a specific TR B The connection sequence of V sequence. Codon diversification was performed using the same method as described in Example 2. Table 1 shows the connection sequence of codon diversification derived from mouse TRAV gene. Table 2 shows the connection sequence of codon diversification derived from mouse TRBV gene. In Tables 1 and 2, the gene name and accession number of each V gene are shown in the first column, and the corresponding connection sequence is shown in the second column.

[0182] Table 1. Junction sequences derived from mouse TRAV gene

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] Table 2. Junction sequences derived from mouse TRBV genes

[0189]

[0190]

[0191] In the initial design, ConA# and ConB# sequences were designed based on the original TRAV or TRBV sequence. As used herein, the symbol # represents the digital ID of the TRAV or TRBV gene. The hybridization yield of each ConX# and each ConX#* was then calculated as a baseline ( Fig. 10A and Fig. 10B ). Fig. 10A Shown are the hybridization yields of junction sequences designed based on the original TRAV sequence without codon diversification (ConA# vs. ConA#*). Fig. 10B The hybridization yields of the junction sequences designed based on the original TRBV sequence without codon diversification (ConB# vs. ConB#*) are shown. During the codon diversification process, the codon selection of the last approximately 60 bases of some TRXV#_GLs was randomized, and ConX# sequences that allowed specific hybridization were selected. Next, the hybridization yields of each ConX# vs. each ConX#* of the sequence group using codon diversification were calculated to observe whether the codon diversification was successful ( Fig.11A and Fig. 11B ). Fig.11AHybridization yields of codon-diversified junction sequences (ConA# vs. ConA#*) are shown. Fig. 11B Hybridization yields of codon-diversified junction sequences (ConB# vs. ConB#*) are shown.

[0192] Example 4. Linking sequences with arbitrary sequences

[0193] Table 3 provides the Figure 7 The scheme described in can be used as any sequence of the linker sequence joining the CDR3-J polynucleotide and the specified V gene germline polynucleotide.

[0194] Table 3. Connection sequences with arbitrary sequences

[0195]

[0196]

[0197]

[0198]

[0199] Example 5. Characterization of assembled TCR genes using next generation sequencing

[0200] A library of nucleic acid sequences encoding paired TCRs was prepared using the methods described herein (e.g., Example 1 with some modifications). Reference sequences encoding naturally paired TCRs were obtained from publicly available libraries. 553 reference sequences were selected to illustrate in this embodiment. In this embodiment, nucleic acid sequences encoding CDR3-Jα (or CDR3-Jα fragments) and nucleic acid sequences encoding CDR3-Jβ (or CDR3-Jβ fragments) were synthesized separately. Alternatively, paired CDR3-Jα and CDR3-Jβ can be synthesized together on one fragment.

[0201] 553 CDR3-Jα fragments and 553 CDR3-Jβ fragments are synthesized and connected (e.g., by connection, overlapping PCR, etc.) together to produce a paired CDR3-Jα-CDR3-Jβ fragment library. In order to ensure that CDR3-Jα is connected to the naturally paired CDR3-Jβ, an arbitrary connection sequence is synthesized on each CDR3-Jα, and the arbitrary connection sequence is designed to minimize cross-hybridization with other arbitrary connection sequences in the CDR3-Jα fragment library. The complementary sequence of the arbitrary connection sequence is synthesized on the naturally paired CDR3-Jβ. Next, the TRAV fragment library (pre-synthesized according to the reference sequence) is connected to the paired CDR3-Jα-CDR3-Jβ fragment to produce a TRAV-CDR3-Jα-CDR3-Jβ fragment library, each fragment comprising a TRAV sequence connected to its homologous CDR3-Jα. Next, the TRBC1 sequence was attached to the downstream of the TRAV-CDR3-Jα-CDR3-Jβ fragment to form the TRAV-CDR3-Jα-CDR3-Jβ-TRBC1 fragment. These fragments were circularized and relinearized by cutting immediately upstream of CDR3-Jβ, thereby forming CDR3-Jβ-TRBC1-TRAV-CDR3-Jα fragments. TRBC1 and TRAV fragments were designed in a way that the in-frame self-cutting P2A sequence connected TRBC1 and TRAV. Next, the TRBV fragment library (pre-synthesized according to the reference sequence) was connected to the CDR3-Jβ-TRBC1-TRA V-CDR3-Jα fragment to produce TRBV-CDR3-Jβ-TRBC1-TRAV-CDR3-Jα, which was subjected to next generation sequencing (NGS) to evaluate the abundance of the clones and the connection accuracy of the clones. Here, each clone in the NGS data refers to a unique sequence. Since 553 reference sequences were used in this example, there were a total of 553 clones in the NGS data. For the data analysis described herein, CDR3-Jα sequences were used to represent clones.

[0202] Fig.12 The accuracy and abundance of each clone after generating paired CDR3-Jα-CDR3-Jβ fragments are shown. Each data point corresponds to a clone of a CDR3-Jα-CDR3-Jβ fragment. Accuracy refers to the fraction of CDR3-Jα fragments connected to homologous CDR3-Jβ fragments. For each CDR-Jα, accuracy can be calculated by dividing the number of correctly connected CDR3-Jβ fragments by the total number of connected CDR3-Jβ fragments. Abundance refers to the fraction of each clone in the total clone library, which can be calculated by dividing the total number of reads of the clone by the total number of reads of all clones. The data show that 497 of 553 clones have an accuracy higher than 95% and an abundance higher than 0.1 / 553, as shown in the box.

[0203] Fig.13 The accuracy and abundance of each clone after generating the TRAV-CDR3-Jα-CDR3-Jβ fragment are shown. Each data point corresponds to a clone of the TRAV-CDR3-Jα-CDR3-Jβ fragment. Accuracy refers to the fraction of CDR3-Jα-CDR3-Jβ fragments connected to the homologous TRAV fragment. For each CDR3-Jα-CDR3-Jβ, the accuracy can be calculated by dividing the number of correctly connected TRAV fragments by the total number of connected TRAV fragments. Abundance refers to the fraction of each clone in the total clone library, which can be calculated by dividing the total number of reads of the clone by the total number of reads of all clones. The data show that 523 of the 553 clones have an accuracy higher than 95% and an abundance higher than 0.1 / 553, as shown in the box.

[0204] Fig.14 A heat map mapping each TRAV to each clone in the library is shown. The clone numbers are sorted according to their cognate TRAV gene names. The data show that for each clone, the majority of reads had the correct TRAV sequence, indicating high accuracy when joining the CDR3-Jα-CDR3-Jβ segments with their cognate TRAV segments.

[0205] Fig.15 The generation of TRAV-CDR3-Jα-CDR3-Jβ fragments (e.g., Fig.15 The abundance of each clone after TRAV addition in ( ) relative to the abundance after generation of CDR3-Jα-CDR3-Jβ fragments. The data suggest that the overall bias is primarily due to the bias during the joining of CDR3-Jα and CDR3-Jβ fragments. This bias can be reduced or avoided by directly synthesizing paired CDR3-Jα-CDR3-Jβ fragments.

[0206] Fig.16 The accuracy and abundance of each clone after generating the TRBV-CDR3-Jβ-TRBC1-TRAV-CDR3-Jα fragment are shown. Each data point corresponds to a clone of a TRBV-CDR3-Jβ-TRBC1-TRAV-CDR3-Jα fragment. Accuracy refers to the fraction of CDR3-Jβ-TRBC1-TRAV-CDR3-Jα fragments connected to homologous TRBV fragments. For each CDR3-Jβ-TRBC1-TRAV-CDR3-Jα, the accuracy can be calculated by dividing the number of correctly connected TRBV fragments by the total number of connected TRBV fragments. Abundance refers to the fraction of each clone in the total clone library, which can be calculated by dividing the total number of reads of the clone by the total number of reads of all clones. The data show that 514 of 553 clones have an accuracy higher than 95% and an abundance higher than 0.1 / 553, as shown in the box.

[0207] Fig.17 A heat map mapping each TRBV to each clone in the library is shown. The clone numbers are sorted according to their cognate TRBV gene names. The data show that for each clone, most reads had the correct TRBV sequence, indicating high accuracy when joining the CDR3-Jβ-TRBC1-TRAV-CDR3-Jα fragments with their cognate TRBV fragments.

[0208] Fig.18 The overall accuracy and abundance of each clone after generating the TRBV-CDR3-Jβ-TRBC1-TRAV-CDR3-Jα fragment are shown. The overall accuracy of each clone was calculated by multiplying Fig.12 , Fig.13 and Fig.16 Accuracy at each step is shown. Abundance was calculated by dividing the total number of reads of that clone by the total number of reads of all clones.

[0209] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many changes, variations and substitutions may be conceived by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used.

[0210] Implementation paragraph

[0211] [1] A method for producing a nucleic acid molecule encoding a T cell receptor (TCR) chain or a portion thereof, comprising: (a) providing at least one nucleic acid molecule comprising a sequence encoding a CDR3 of a TCR chain; (b) providing a plurality of nucleic acid molecules, each of the plurality of nucleic acid molecules comprising a sequence derived from a TCR V gene, wherein the plurality of nucleic acid molecules comprise at least two different sequences derived from at least two different TCR V genes; and (c) contacting the at least one nucleic acid molecule of (a) with the plurality of nucleic acid molecules of (b) in the same compartment, wherein the at least one nucleic acid molecule of (a) is capable of connecting with a nucleic acid molecule in the plurality of nucleic acid molecules to produce a third nucleic acid molecule comprising a sequence encoding the CDR3 and a sequence derived from one of the at least two different TCR V genes, thereby producing the nucleic acid molecule encoding the TCR chain or a portion thereof.

[0212] [2] The method of paragraph [1], wherein the at least one nucleic acid molecule comprises a first plurality of nucleic acid molecules, wherein each nucleic acid molecule in the first plurality of nucleic acid molecules comprises a sequence encoding a CDR3 of a TCR chain.

[0213] [3] A method as described in paragraph [1] or [2], wherein the at least one nucleic acid molecule of (a) is capable of specifically linking to a nucleic acid molecule among the plurality of nucleic acid molecules, wherein the nucleic acid molecule among the plurality of nucleic acid molecules comprises a sequence derived from any single given TCR V gene among the at least two different TCR V genes.

[0214] [4] The method of paragraph [1], wherein the at least one nucleic acid molecule further comprises the J region of the TCR chain.

[0215] [5] The method of paragraph [2], wherein each nucleic acid molecule in the first plurality of nucleic acid molecules further comprises a J region of a TCR chain.

[0216] [6] The method of any one of paragraphs [1]-[5], wherein the at least two TCR V genes are human TCR V genes or mouse TCR V genes.

[0217] [7] The method of any one of paragraphs [1] to [6], wherein the at least two TCR V genes are selected from human TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV7, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8-6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV13-2, TRAV14 , TRAV16, TRAV17, TRAV18, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23, TRAV24, TRAV25, TRAV26-1, TRAV26-2, TRAV27, TRAV29, TRAV30, TRAV34, TRAV35, TRAV36, TRAV38-1, TRAV38-2, TRAV39, TRAV40 and TRAV41.

[0218] [8] The method of any one of paragraphs [1] to [6], wherein the at least two TCR V genes are selected from human TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV4-3, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV5-8, TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV6-9, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-8, A group consisting of TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1 and TRBV30.

[0219] [9] The method of any one of paragraphs [1]-[8], wherein each of the multiple sequences derived from the at least two different TCR V genes comprises a sequence encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and / or FR3.

[0220]

[10] The method of any one of paragraphs [1]-[9], wherein the TCR chain is a TCR α chain, a TCR β chain, a TCR γ chain or a TCR δ chain.

[0221]

[11] The method of any of paragraphs [1]-

[10] , wherein the at least one nucleic acid molecule further comprises an additional sequence encoding an additional CDR3 of an additional TCR chain.

[0222]

[12] The method of paragraph

[11] , wherein the at least one nucleic acid molecule comprises an additional J region of the additional TCR chain.

[0223]

[13] The method of paragraph

[11] or

[12] , wherein the sequence encoding the CDR3 and the additional sequence encoding the additional CDR3 are separated by at most 100 nucleotides.

[0224]

[14] The method of any one of paragraphs

[11] -

[13] , wherein the TCR chain and the additional TCR chain are a cognate pair of TCR chains.

[0225]

[15] A method as described in any of paragraphs [1]-

[14] , wherein the at least one nucleic acid molecule comprises a linker sequence, which is capable of linking the at least one nucleic acid molecule to a nucleic acid molecule in the plurality of nucleic acid molecules to produce the third nucleic acid molecule.

[0226]

[16] The method of paragraph

[15] , wherein the at least one nucleic acid molecule and the nucleic acid molecules among the plurality of nucleic acid molecules encode a functional TCR chain or a portion thereof.

[0227]

[17] The method of paragraph

[15] or

[16] , wherein a nucleic acid molecule among the plurality of nucleic acid molecules comprises an anti-ligation sequence, wherein the anti-ligation sequence is complementary to the ligation sequence of the at least one nucleic acid molecule of (a).

[0228]

[18] The method of any one of paragraphs [1]-

[17] , further comprising connecting the at least one nucleic acid molecule of (a) and the nucleic acid molecules of the plurality of nucleic acid molecules of (b).

[0229]

[19] The method of paragraph

[18] , wherein the ligation comprises hybridizing the at least one nucleic acid molecule of (a) with a nucleic acid molecule among the plurality of nucleic acid molecules of (b).

[0230]

[20] The method of paragraph

[19] , wherein the hybridization comprises hybridizing the connecting sequence of the at least one nucleic acid molecule of (a) with the anti-connection sequence of a nucleic acid molecule among the plurality of nucleic acid molecules of (b).

[0231]

[21] A method as described in any of paragraphs

[18] -

[20] , which also includes (i) using the at least one nucleic acid molecule of (a) as a template to extend the free 3' end of a nucleic acid molecule among the multiple nucleic acid molecules, and / or (ii) using a nucleic acid molecule among the multiple nucleic acid molecules as a template to extend the free 3' end of the at least one nucleic acid molecule of (a) to produce the third nucleic acid molecule.

[0232]

[22] The method of any one of paragraphs [1]-

[21] , further comprising connecting the at least one nucleic acid molecule of (a) to a nucleic acid molecule among the plurality of nucleic acid molecules of (b).

[0233]

[23] The method of any one of paragraphs [1]-

[22] , further comprising contacting the third nucleic acid molecule with a restriction enzyme to produce sticky ends.

[0234]

[24] The method of any one of paragraphs [1]-

[23] , further comprising contacting the third nucleic acid molecule with an additional nucleic acid molecule.

[0235]

[25] The method of paragraph

[24] , wherein the additional nucleic acid molecule encodes a constant region of a TCR chain or a portion thereof.

[0236]

[26] The method of paragraph

[24] or

[25] , further comprising linking the third nucleic acid molecule to the additional nucleic acid molecule.

[0237]

[27] The method of any of paragraphs [1]-

[26] , wherein multiple nucleic acid molecules, each encoding a different TCR chain or portion thereof, are produced in the same compartment.

[0238]

[28] The method of paragraph

[27] , wherein at least five different nucleic acid molecules among the plurality of nucleic acid molecules are produced in the same compartment.

[0239]

[29] The method of any one of paragraphs [1]-

[26] , wherein at least ten different nucleic acid molecules among the plurality of nucleic acid molecules are produced in the same compartment.

[0240]

[30] The method of any one of paragraphs [1]-

[29] , wherein the same compartment is a well, a tube or a droplet.

[0241]

[31] The method of any of paragraphs [1]-

[30] , wherein at least one of the nucleic acid molecules comprises a unique barcode.

[0242]

[32] The method of paragraph

[31] , wherein the unique barcode is a primer binding site.

[0243]

[33] The method of any of paragraphs

[15] -

[30] , wherein the ligation sequence comprises a unique barcode.

[0244]

[34] The method of paragraph

[33] , wherein the unique barcode is a primer binding site.

[0245]

[35] A composition comprising

[0246] (a) a plurality of nucleic acid molecules, wherein each nucleic acid molecule in the plurality comprises a sequence derived from a T cell receptor (TCR) V gene and does not comprise a CDR3 sequence, wherein a first nucleic acid molecule in the plurality comprises a first anti-ligation sequence and a second nucleic acid molecule in the plurality comprises a second anti-ligation sequence, wherein the first anti-ligation sequence is different from the second anti-ligation sequence, and wherein the sequences derived from the TCR V gene of the first nucleic acid molecule and the second nucleic acid molecule are derived from different TCR V genes; and

[0247] (b) at least one nucleic acid molecule comprising a sequence encoding a CDR3 of a TCR chain, wherein the at least one nucleic acid molecule further comprises a first joining sequence complementary to the first anti-joining sequence.

[0248]

[36] The composition of paragraph

[35] , wherein the composition is a liquid composition.

[0249]

[37] The composition of paragraph

[35] or

[36] , wherein the multiple nucleic acid molecules of (a) and the at least one nucleic acid molecule of (b) are in the same compartment.

[0250]

[38] A composition as described in any of paragraphs

[35] -

[37] , wherein the sequence derived from the TCR V gene comprises at least ten nucleotides of the TCR V gene.

[0251]

[39] The composition of any one of paragraphs

[35] -

[38] , wherein the TCR V gene is a TRAV gene, a TRBV gene, a TRGV gene or a TRDV gene.

[0252]

[40] A composition as described in any of paragraphs

[35] -

[39] , wherein the sequence derived from the TCR V gene comprises a sequence encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and / or FR3.

[0253]

[41] The composition of any of paragraphs

[35] -

[40] , wherein the at least one nucleic acid molecule further comprises the J region of the TCR chain.

[0254]

[42] The composition of any of paragraphs

[35] -

[41] , wherein the at least one nucleic acid molecule further comprises an additional sequence encoding an additional CDR3 of an additional TCR chain.

[0255]

[43] The composition of paragraph

[42] , wherein the at least one nucleic acid molecule further comprises an additional J region of the additional TCR chain.

[0256]

[44] The composition of paragraph

[42] or

[43] , wherein the sequence encoding CDR3 and the additional sequence encoding CDR3 are separated by at most 100 nucleotides.

[0257]

[45] The composition of any of paragraphs

[42] -

[44] , wherein the TCR chain and the additional TCR chain are a cognate pair of TCR chains.

[0258]

[46] A composition as described in any of paragraphs

[35] -

[45] , wherein the at least one nucleic acid molecule of (b) comprises a first plurality of nucleic acid molecules, and wherein each nucleic acid molecule in the first plurality of nucleic acid molecules comprises a sequence encoding a CDR3 of a TCR chain.

[0259]

[47] The composition of paragraph

[46] , wherein each nucleic acid molecule in the first plurality of nucleic acid molecules encodes a different CDR3 of a different TCR chain.

[0260]

[48] ​​A composition as described in paragraph

[46] or

[47] , wherein each nucleic acid molecule in the first plurality of nucleic acid molecules comprises a different linker sequence, and the different linker sequence is capable of specifically linking to a nucleic acid molecule in the plurality of nucleic acid molecules comprising a sequence derived from any single given TCR V gene.

[0261]

[49] The composition of any of paragraphs

[35] -

[48] , wherein the first anti-linking sequence or the second anti-linking sequence comprises a TCR V gene sequence.

[0262]

[50] The composition of paragraph

[49] , wherein the TCR V gene sequence comprises at least three nucleotides of the TCR V gene adjacent to the sequence encoding CDR3 in the rearranged gene.

[0263]

[51] The composition of any of paragraphs

[35] -

[50] , wherein the first anti-ligation sequence or the second anti-ligation sequence comprises a predetermined sequence.

[0264]

[52] The composition of any of paragraphs

[35] -

[51] , wherein the first linking sequence hybridizes to the first anti-linking sequence.

[0265]

[53] The composition of any of paragraphs

[35] -

[52] , wherein the at least one nucleic acid molecule of (b) comprises a unique barcode.

[0266]

[54] The composition of paragraph

[53] , wherein the unique barcode is a primer binding site.

[0267]

[55] The composition of any of paragraphs

[35] -

[52] , wherein the first ligated sequence of the at least one nucleic acid molecule comprises a unique barcode.

[0268]

[56] The composition of paragraph

[55] , wherein the unique barcode is a primer binding site.

[0269]

[57] A method for producing multiple nucleic acid molecules, comprising: (a) providing a first plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules comprise sequences encoding a first CDR3 of a first T cell receptor (TCR) chain and a second CDR3 of a second TCR chain, wherein the first CDR3 and the second CDR3 are derived from a homologous pair of TCR chains; (b) providing a second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the second plurality of nucleic acid molecules comprise sequences derived from TCR V genes, wherein the nucleic acid molecules do not comprise sequences encoding a constant domain; and (c) contacting the first plurality of nucleic acid molecules with the second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules are linked to the nucleic acid molecules in the second plurality of nucleic acid molecules to form a nucleic acid molecule comprising the sequences encoding the first CDR3 and the second CDR3 and the sequences derived from the TCR V genes, wherein the sequences encoding the first CDR3 and the second CDR3 and the TCR V genes are derived from a homologous pair of TCR chains.

[0270]

[58] The method of paragraph

[57] , wherein each nucleic acid molecule of the first plurality of nucleic acid molecules comprises a sequence encoding a different first CDR3 of the first TCR chain and / or a different CDR3 of the second TCR chain.

[0271]

[59] The method of paragraph

[57] or

[58] , wherein each nucleic acid molecule in the second plurality of nucleic acid molecules comprises a sequence derived from a different TCR V gene.

[0272]

[60] The method of any of paragraphs

[57] -

[59] , wherein the first plurality of nucleic acid molecules and the second plurality of nucleic acid molecules are contacted in the same compartment.

[0273]

[61] A method as described in any of paragraphs

[57] -

[60] , wherein the nucleic acid molecules in the first plurality of nucleic acid molecules further comprise a linker sequence, wherein the linker sequence links the nucleic acid molecules in the first plurality of nucleic acid molecules to the nucleic acid molecules in the second plurality of nucleic acid molecules.

[0274]

[62] The method of paragraph

[61] , wherein the nucleic acid molecules in the second plurality of nucleic acid molecules further comprise an anti-ligation sequence, wherein the anti-ligation sequence is complementary to the ligation sequence.

[0275]

[63] The method of paragraph

[62] , wherein the linking sequence hybridizes with the anti-linking sequence to link a nucleic acid molecule in the first plurality of nucleic acid molecules to a nucleic acid molecule in the second plurality of nucleic acid molecules.

[0276]

[64] A method as described in any of paragraphs

[58] -

[63] , wherein the connecting sequence is codon diversified so that the connecting sequence of a nucleic acid molecule in the first plurality of nucleic acid molecules is different from other connecting sequences of other nucleic acid molecules in the first plurality of nucleic acid molecules.

[0277]

[65] The method of any one of paragraphs

[57] -

[64] , wherein the nucleic acid molecules in the first plurality of nucleic acid molecules further comprise the first J region of the first TCR chain and / or the second J region of the second TCR chain.

[0278]

[66] The method of any of paragraphs

[57] -

[65] , wherein (i) the first TCR chain is a TCR α chain and the second TCR chain is a TCR β chain, or (ii) the first TCR chain is a TCR γ chain and the second TCR chain is a TCR δ chain.

[0279]

[67] The method of any one of paragraphs

[57] -

[66] , wherein the TCR V gene is a TRAV gene, a TRBV gene, a TRGV gene or a TRDV gene.

[0280]

[68] The method of any of paragraphs

[57] -

[67] , wherein the nucleic acid molecules in the second plurality of nucleic acid molecules are double-stranded nucleic acid molecules.

[0281]

[69] The method of any of paragraphs

[57] -

[68] , wherein the nucleic acid molecules in the second plurality of nucleic acid molecules further comprise a sequence encoding a portion of a self-cleaving peptide.

[0282]

[70] The method of any of paragraphs

[62] -

[69] , wherein the anti-ligation sequence is an overhang of a nucleic acid molecule in the second plurality of nucleic acid molecules.

[0283]

[71] The method of any one of paragraphs

[62] -

[70] , wherein the linking sequence or the anti-linking sequence is at least three nucleotides in length.

[0284]

[72] The method as described in any of paragraphs

[63] -

[71] , which also includes (i) extending the 3' end of the nucleic acid molecule in the first plurality of nucleic acid molecules that hybridizes with the nucleic acid molecule in the second plurality of nucleic acid molecules and / or (ii) extending the 3' end of the nucleic acid molecule in the second plurality of nucleic acid molecules that hybridizes with the nucleic acid molecule in the first plurality of nucleic acid molecules.

[0285]

[73] The method of any of paragraphs

[57] -

[72] , further comprising connecting a nucleic acid molecule from the first plurality of nucleic acid molecules to a nucleic acid molecule from the second plurality of nucleic acid molecules.

[0286]

[74] The method as described in any of paragraphs

[57] -

[73] , which also includes contacting the nucleic acid molecule comprising the sequences encoding the first CDR3 and the second CDR3 and the sequence derived from the TCR V gene with a restriction enzyme to produce sticky ends.

[0287]

[75] The method as described in any of paragraphs

[57] -

[74] , contacting the nucleic acid molecule comprising the sequence encoding the first CDR3 and the second CDR3 and the sequence derived from the TCR V gene with an additional nucleic acid molecule comprising a sequence encoding a constant region or a portion thereof.

[0288]

[76] The method as described in paragraph

[74] or

[75] further includes connecting the nucleic acid molecule comprising the sequences encoding the first CDR3 and the second CDR3 and the sequence derived from the TCR V gene to an additional nucleic acid molecule via sticky ends.

[0289]

[77] The method of any of paragraphs

[57] -

[76] , wherein the sequence encoding the first CDR3 and the sequence encoding the second CDR3 are separated by at most 100 nucleotides.

[0290]

[78] The method of any of paragraphs

[57] -

[77] , wherein the sequence derived from the TCR V gene comprises sequences encoding FR1, CDR1, FR2, CDR2 and FR3.

[0291]

[79] The method of any one of paragraphs

[57] -

[77] , wherein the sequence derived from the TCR V gene comprises sequences encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and FR3.

[0292]

[80] A composition comprising: (a) a first plurality of nucleic acid molecules, wherein each nucleic acid molecule in the first plurality of nucleic acid molecules comprises a sequence encoding a first CDR3 of a first T cell receptor (TCR) chain and a second CDR3 of a second TCR chain, wherein the first CDR3 and the second CDR3 are from a cognate pair of TCR chains; and (b) a second plurality of nucleic acid molecules, wherein each nucleic acid molecule in the second plurality of nucleic acid molecules comprises a sequence derived from a TCR V gene, and wherein each nucleic acid molecule in the second plurality of nucleic acid molecules does not comprise a sequence encoding the first CDR3 and the second CDR3;

[0293] Wherein (i) each nucleic acid molecule of the first plurality of nucleic acid molecules comprises a sequence encoding a different first CDR3 and / or second CDR3, and / or (ii) each nucleic acid molecule of the second plurality of nucleic acid molecules comprises a sequence derived from a different TCR V gene.

[0294]

[81] A composition as described in paragraph

[80] , wherein each nucleic acid molecule in the first plurality of nucleic acid molecules further comprises a linker sequence, wherein a given linker sequence can be used to link a given nucleic acid molecule in the first plurality of nucleic acid molecules to a given nucleic acid molecule in the second plurality of nucleic acid molecules.

[0295]

[82] The composition of paragraph

[80] or

[81] , wherein each nucleic acid molecule in the second plurality of nucleic acid molecules further comprises an anti-ligation sequence that is complementary to the ligation sequence.

[0296]

[83] A composition as described in paragraph

[81] or

[82] , wherein the linker sequence is codon-diverse such that a given linker sequence of a given nucleic acid molecule in the first plurality of nucleic acid molecules is different from other linker sequences of other nucleic acid molecules in the first plurality of nucleic acid molecules.

[0297]

[84] A composition as described in any of paragraphs

[81] -

[83] , wherein the linker sequence encodes an amino acid sequence.

[0298]

[85] The composition of paragraph

[84] , wherein the linker sequence is in frame with the sequence encoding the first CDR3 of the first TCR chain and the second CDR3 of the second TCR chain.

[0299]

[86] A composition as described in any of paragraphs

[81] -

[85] , wherein the linker sequence comprises at least three nucleotides.

[0300]

[87] The composition of paragraph

[86] , wherein the linker sequence comprises at least three nucleotides of the TCR V gene adjacent to a sequence encoding the first CDR3 of the first TCR chain or the second CDR3 of the second TCR chain in the rearranged gene.

[0301]

[88] A composition as described in any of paragraphs

[84] -

[87] , wherein the given amino acid sequence encoded by the given linked sequence is identical or substantially identical to at least one other amino acid sequence encoded by at least one other linked sequence.

[0302]

[89] A composition as described in any of paragraphs

[84] -

[87] , wherein the given amino acid sequence encoded by the given linker sequence is different from other amino acid sequences encoded by other linker sequences.

[0303]

[90] The composition of any of paragraphs

[80] -

[89] , wherein each of the first plurality of nucleic acid molecules further comprises the first J region of the first TCR chain and / or the second J region of the second TCR chain.

[0304]

[91] The composition of any of paragraphs

[80] -

[90] , wherein the composition is a liquid composition.

[0305]

[92] The composition of any of paragraphs

[80] -

[91] , wherein the first plurality of nucleic acid molecules and the second plurality of nucleic acid molecules are in the same compartment.

[0306]

[93] The composition of any of paragraphs

[81] -

[92] , wherein a given nucleic acid molecule in the first plurality of nucleic acid molecules is linked to a given nucleic acid molecule in the second plurality of nucleic acid molecules via the given linking sequence.

[0307]

[94] The composition of paragraph

[93] , wherein a given nucleic acid molecule in the first plurality of nucleic acid molecules hybridizes to a given nucleic acid molecule in the second plurality of nucleic acid molecules via the given linker sequence that hybridizes to a given anti-linker sequence.

[0308]

[95] The composition of any of paragraphs

[80] -

[94] , wherein the sequence encoding the first CDR3 and the sequence encoding the second CDR3 are separated by at most 100 nucleotides.

[0309]

[96] The composition of any of paragraphs

[80] -

[95] , wherein the sequence derived from the TCR V gene comprises sequences encoding FR1, CDR1, FR2, CDR2 and FR3.

[0310]

[97] A composition as described in any of paragraphs

[80] -

[95] , wherein the sequence derived from the TCR V gene comprises a sequence encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and FR3.

[0311]

[98] The composition of any of paragraphs

[80] -

[97] , wherein each nucleic acid molecule in the first plurality of nucleic acid molecules or the second plurality of molecules is chemically synthesized.

[0312]

[99] The composition of any of paragraphs

[80] -

[98] , wherein each nucleic acid molecule in the first plurality of nucleic acid molecules is at most about 250 nucleotides in length.

[0313]

[100] A composition comprising a plurality of nucleic acid molecules, each of the plurality of nucleic acid molecules comprising a sequence derived from a T cell receptor (TCR) V gene, wherein the plurality of nucleic acid molecules comprises a first nucleic acid molecule having a first connecting sequence and a second nucleic acid molecule having a second connecting sequence, wherein the first connecting sequence is different from the second connecting sequence.

[0314]

[101] The composition of paragraph

[100] , wherein each of the plurality of nucleic acid molecules comprises a sequence derived from a different TCR V gene.

[0315]

[102] The composition of paragraph

[100] or

[101] , wherein each nucleic acid molecule in the plurality of nucleic acid molecules comprises a different linking sequence.

[0316]

[103] A composition as described in any of paragraphs

[100] -

[102] , wherein each nucleic acid molecule in the plurality of nucleic acid molecules does not contain a sequence encoding CDR3 of a TCR chain.

[0317]

[104] A composition as described in any of paragraphs

[100] -

[103] , wherein each nucleic acid molecule in the plurality of nucleic acid molecules does not contain a sequence encoding a constant domain of a TCR chain.

[0318]

[105] A composition as described in any of paragraphs

[100] -

[104] , wherein the sequence derived from the TCR V gene comprises at least ten nucleotides of the TCR V gene.

[0319]

[106] A composition as described in any of paragraphs

[100] -

[105] , wherein the TCR V gene is a TRAV gene, a TRBV gene, a TRGV gene or a TRDV gene.

[0320]

[107] A composition comprising a plurality of nucleic acid molecules, each of the plurality of nucleic acid molecules encoding a CDR3 of a T cell receptor (TCR) chain, wherein a first nucleic acid molecule in the plurality comprises a first linker sequence and a second nucleic acid molecule in the plurality comprises a second linker sequence, wherein the first linker sequence is different from the second linker sequence.

[0321]

[108] A composition as described in paragraph

[107] , wherein each of the multiple nucleic acid molecules further comprises a J region of a TCR chain.

[0322]

[109] The composition of paragraph

[107] , wherein each of the plurality of nucleic acid molecules encodes a first CDR3 of a first TCR chain and a second CDR3 of a second TCR chain.

[0323]

[110] A composition as described in paragraph

[109] , wherein each of the multiple nucleic acid molecules further comprises a first J region of a first TCR chain and a second J region of a second TCR chain.

[0324]

[111] A composition as described in any of paragraphs

[107] -

[110] , wherein each nucleic acid molecule in the plurality of nucleic acid molecules encodes a different CDR3 of a different TCR chain.

[0325]

[112] A composition as described in any of paragraphs

[107] -

[111] , wherein each nucleic acid molecule in the plurality of nucleic acid molecules comprises a different linking sequence.

[0326]

[113] A composition as described in any of paragraphs

[107] -

[112] , wherein each nucleic acid molecule in the plurality of nucleic acid molecules does not contain more than 200 nucleotides of a TCR V gene.

[0327]

[114] A composition as described in any of paragraphs

[107] -

[113] , wherein each nucleic acid molecule in the plurality of nucleic acid molecules does not contain a sequence encoding a constant domain of a TCR chain.

[0328]

[115] A composition as described in any of paragraphs

[100] -

[114] , wherein the first connecting sequence or the second connecting sequence comprises a sequence derived from a TCR V gene.

[0329]

[116] A composition as described in paragraph

[115] , wherein the sequence derived from the TCR V gene comprises at least three nucleotides of the TCR V gene adjacent to the sequence encoding CDR3 in the rearranged gene.

[0330]

[117] The composition of any of paragraphs

[100] -

[116] , wherein the first linker sequence or the second linker sequence comprises a predetermined sequence.

[0331]

[118] A composition as described in any of paragraphs

[107] -

[114] , wherein the first connecting sequence or the second connecting sequence comprises a sequence complementary to a TCR V gene sequence.

[0332]

[119] A composition as described in any of paragraphs

[107] -

[114] and

[118] , wherein the composition further comprises a second plurality of nucleic acid molecules, each of the second plurality of nucleic acid molecules comprising a sequence derived from a TCR V gene.

[0333]

[120] The composition of paragraph

[119] , wherein the first nucleic acid molecule in the second plurality comprises a first anti-ligation sequence that is complementary to the first ligation sequence.

[0334]

[121] The composition of paragraph

[119] or

[120] , wherein the second nucleic acid molecule in the second plurality comprises a second anti-ligation sequence that is complementary to the second ligation sequence.

[0335]

[122] The composition of paragraph

[120] or

[121] , wherein the first anti-ligation sequence of the first nucleic acid molecule in the second plurality is linked to the first ligation sequence of the first nucleic acid molecule in the first plurality.

[0336]

[123] A composition as described in paragraph

[121] or

[122] , wherein the second anti-ligation sequence of the second nucleic acid molecule in the second plurality is linked to the second ligation sequence of the second nucleic acid molecule in the first plurality.

[0337]

[124] A composition comprising a plurality of nucleic acid molecules, each comprising a sequence of at least ten amino acids encoding a T cell receptor (TCR) chain, wherein a first nucleic acid molecule of the plurality comprises a first linker sequence and a second nucleic acid molecule of the plurality comprises a second linker sequence, wherein the first linker sequence is different from the second linker sequence, wherein the first linker sequence or the second linker sequence encodes a portion of a TCR chain, and wherein the first linker sequence or the second linker sequence is in frame with the sequence of at least ten amino acids encoding the TCR chain.

[0338]

[125] The composition of paragraph

[124] , wherein the first linker sequence or the second linker sequence comprises at least four consecutive nucleotides of a TCR chain gene and is in frame with the sequence encoding at least ten amino acids of the TCR chain.

[0339]

[126] A composition as described in paragraph

[124] or

[125] , wherein the first linker sequence and the second linker sequence encode at least two consecutive amino acids of the TCR chain.

[0340]

[127] A composition as described in any of paragraphs

[124] -

[126] , wherein the TCR chain of the portion of the TCR chain is identical to the TCR chain encoded by a sequence encoding at least ten amino acids.

[0341]

[128] The composition of paragraph

[124] , wherein each of the plurality of nucleic acid molecules comprises a sequence derived from a TCR V gene.

[0342]

[129] A composition as described in any of paragraphs

[124] -

[128] , wherein each nucleic acid molecule of the plurality of nucleic acid molecules encodes a CDR3 of the TCR chain.

[0343]

[130] A composition as described in paragraph

[129] , wherein each of the multiple nucleic acid molecules further comprises the J region of the TCR chain.

[0344]

[131] The composition of paragraph

[129] , wherein each of the plurality of nucleic acid molecules encodes a first CDR3 of a first TCR chain and a second CDR3 of a second TCR chain.

[0345]

[132] A composition as described in paragraph

[131] , wherein each of the multiple nucleic acid molecules further comprises a first J region of a first TCR chain and a second J region of a second TCR chain.

[0346]

[133] The composition of paragraph

[131] or

[132] , wherein the sequence encoding the first CDR3 and the sequence encoding the second CDR3 are separated by at most 100 nucleotides.

[0347]

[134] A composition as described in any of paragraphs

[124] -

[133] , wherein the first connecting sequence or the second connecting sequence comprises a sequence derived from a TCR V gene.

[0348]

[135] The composition of any of paragraphs

[124] -

[134] , wherein the first linker sequence or the second linker sequence comprises a predetermined sequence.

[0349]

[136] The composition of any of paragraphs

[100] -

[135] , wherein the first linking sequence comprises at least one nucleotide that is different from a nucleotide of the second linking sequence.

[0350]

[137] A composition as described in any of paragraphs

[100] -

[136] , wherein the first linker sequence and the second linker sequence encode the same amino acid sequence.

[0351]

[138] A composition as described in any of paragraphs

[100] -

[136] , wherein the first linker sequence and the second linker sequence encode different amino acid sequences.

[0352]

[139] A method for producing a plurality of nucleic acid molecules, each of the plurality encoding a T cell receptor (TCR) chain or a region thereof, the method comprising: contacting a first plurality of nucleic acid molecules with a second plurality of nucleic acid molecules to produce a third plurality of nucleic acid molecules, the third plurality of nucleic acid molecules comprising at least two different nucleic acid molecules, wherein each of the at least two different nucleic acid molecules has a different sequence encoding a different TCR chain or a region thereof, and wherein the at least two different nucleic acid molecules are produced in the same compartment.

[0353]

[140] The method of paragraph

[139] , wherein each nucleic acid molecule of the first plurality of nucleic acid molecules comprises a sequence encoding the CDR3 of the TCR chain.

[0354]

[141] The method of paragraph

[140] , wherein each nucleic acid molecule of the first plurality of nucleic acid molecules comprises the J region of the TCR chain.

[0355]

[142] The method of any of paragraphs

[139] -

[141] , wherein each nucleic acid molecule of the second plurality of nucleic acid molecules comprises a sequence derived from a TCR V gene of the TCR chain.

[0356]

[143] The method of paragraph

[142] , wherein the TCR V gene is a human TCR V gene.

[0357]

[144] The method of paragraph

[142] or

[143] , wherein the TCR V gene is human TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV7, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8-6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV13-2, TRAV1 4. TRAV16, TRAV17, TRAV18, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23, TRAV24, TRAV25, TRAV26-1, TRAV26-2, TRAV27, TRAV29, TRAV30, TRAV34, TRAV35, TRAV36, TRAV38-1, TRAV38-2, TRAV39, TRAV40 or TRAV41.

[0358]

[145] The method of paragraphs

[142] or

[143] , wherein the TCR V gene is human TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV4-3, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV5-8, TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV6-9, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-8 , TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, T RBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1, or TRBV30.

[0359]

[146] A method as described in any of paragraphs

[139] -

[145] , wherein the sequence derived from the TCR V gene includes sequences encoding FR1, CDR1, FR2, CDR2 and FR3.

[0360]

[147] A method as described in any of paragraphs

[139] -

[145] , wherein the sequence derived from the TCR V gene includes sequences encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and FR3.

[0361]

[148] The method of any one of paragraphs

[139] -

[147] , wherein the TCR chain is a TCR α chain, a TCR β chain, a TCR γ chain or a TCR δ chain.

[0362]

[149] The method of any of paragraphs

[140] -

[148] , wherein each nucleic acid molecule in the first plurality of nucleic acid molecules further comprises an additional sequence encoding an additional CDR3 of an additional TCR chain.

[0363]

[150] The method of paragraph

[149] , wherein each nucleic acid molecule of the first plurality of nucleic acid molecules comprises an additional J region of the additional TCR chain.

[0364]

[151] The method of paragraphs

[149] or

[150] , wherein the TCR chain and the additional TCR chain are a cognate pair of TCR chains.

[0365]

[152] A method as described in any of paragraphs

[139] -

[151] , wherein the nucleic acid molecules in the plurality of nucleic acid molecules encode different TCRs or regions thereof.

[0366]

[153] A method as described in any of paragraphs

[139] -

[152] , wherein a given nucleic acid molecule in the first plurality of nucleic acid molecules comprises a linker sequence, which can be used to link the given nucleic acid molecule in the first plurality of nucleic acid molecules to a given nucleic acid molecule in the second plurality of nucleic acid molecules.

[0367]

[154] The method of paragraph

[153] , wherein a given nucleic acid molecule among the first plurality of nucleic acid molecules and a given nucleic acid molecule among the second plurality of nucleic acid molecules encode a functional TCR chain or a region thereof.

[0368]

[155] A method as described in paragraphs

[153] or

[154] , wherein a given nucleic acid molecule in the second plurality of nucleic acid molecules comprises an anti-ligation sequence, which is complementary to the ligation sequence of the given nucleic acid molecule in the first plurality of nucleic acid molecules.

[0369]

[156] A method as described in any of paragraphs

[153] -

[155] , which also includes connecting a given nucleic acid molecule among the first plurality of nucleic acid molecules to a given nucleic acid molecule among the second plurality of nucleic acid molecules.

[0370]

[157] A method as described in paragraph

[156] , wherein the ligation includes hybridizing a given nucleic acid molecule among the first plurality of nucleic acid molecules with a given nucleic acid molecule among the second plurality of nucleic acid molecules.

[0371]

[158] A method as described in paragraph

[157] , wherein the hybridization includes hybridizing the connecting sequence of a given nucleic acid molecule in the first plurality of nucleic acid molecules with the anti-connection sequence of a given nucleic acid molecule in the second plurality of nucleic acid molecules.

[0372]

[159] A method as described in any of paragraphs

[156] -

[158] , which also includes (i) using a given nucleic acid molecule among the first plurality of nucleic acid molecules as a template to extend the free 3' end of a given nucleic acid molecule among the second plurality of nucleic acid molecules, and / or (ii) using a given nucleic acid molecule among the second plurality of nucleic acid molecules as a template to extend the free 3' end of a nucleic acid molecule among the first plurality of nucleic acid molecules to produce a nucleic acid molecule among the third plurality of nucleic acid molecules.

[0373]

[160] The method of any of paragraphs

[139] -

[159] , further comprising linking a given nucleic acid molecule among the first plurality of nucleic acid molecules to a given nucleic acid molecule among the second plurality of nucleic acid molecules.

[0374]

[161] The method of any of paragraphs

[139] -

[160] , further comprising contacting the nucleic acid molecules in the third plurality of nucleic acid molecules with a restriction enzyme to produce sticky ends.

[0375]

[162] The method of any of paragraphs

[139] -

[161] , further comprising contacting a nucleic acid molecule in the third plurality of nucleic acid molecules with an additional nucleic acid molecule.

[0376]

[163] The method of paragraph

[162] , wherein the additional nucleic acid molecule encodes a constant region or a portion thereof of a TCR chain.

[0377]

[164] A method as described in paragraphs

[162] or

[163] , which also includes connecting the nucleic acid molecules in the third plurality of nucleic acid molecules to the additional nucleic acid molecules.

[0378]

[165] The method of any of paragraphs

[139] -

[164] , wherein at least five different nucleic acid molecules of the third plurality of nucleic acid molecules are produced in the same compartment.

[0379]

[166] The method of any of paragraphs

[139] -

[165] , wherein at least ten different nucleic acid molecules of the third plurality of nucleic acid molecules are produced in the same compartment.

[0380]

[167] The method of any of paragraphs

[139] -

[166] , wherein the same compartment is a well, a tube or a droplet.

[0381]

[168] A method for producing multiple nucleic acid molecules, comprising: (a) providing a first plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules comprise sequences encoding a first CDR3 of a first T cell receptor (TCR) chain and a second CDR3 of a second TCR chain, wherein the first CDR3 and the second CDR3 are derived from a homologous pair of TCR chains; (b) providing a second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the second plurality of nucleic acid molecules comprise sequences derived from TCR V genes; and (c) contacting the first plurality of nucleic acid molecules with the second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules are linked to the nucleic acid molecules in the second plurality of nucleic acid molecules to form a linear nucleic acid molecule comprising the sequences encoding the first CDR3 and the second CDR3 and the sequences derived from the TCR V genes, wherein the sequences encoding the first CDR3 and the second CDR3 and the TCR V genes are derived from a homologous pair of TCR chains.

[0382]

[169] A method for producing a plurality of nucleic acid molecules, comprising: (a) providing a first plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules comprise (i) a synthetic sequence encoding a first CDR3 of a first T cell receptor (TCR) chain and a second CDR3 of a second TCR chain, and (ii) a synthetic sequence encoding a third CDR3 of a third T cell receptor (TCR) chain and a fourth CDR3 of a fourth TCR chain, wherein the first CDR3 and the second CDR3 are from a first homologous pair of TCR chains, and wherein the third CDR3 and the fourth CDR3 are from a second homologous pair of TCR chains; (b) providing a second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the second plurality of nucleic acid molecules comprise a sequence derived from a TCR V gene; and (c) contacting the first plurality of nucleic acid molecules with the second plurality of nucleic acid molecules, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules are linked to the nucleic acid molecules in the second plurality of nucleic acid molecules to form a nucleic acid molecule comprising the sequences encoding the first CDR3 and the second CDR3 and the sequence derived from the TCR V gene, wherein the sequences encoding the first CDR3 and the second CDR3 and the TCR The V genes are derived from cognate pairs of the TCR chains.

[0383]

[170] A method for identifying the sequence of a naturally paired T cell receptor (TCR) in a tissue sample from a subject, comprising: (a) identifying one or more pairing sequences of one or more naturally paired TCRs in a sample containing a plurality of peripheral T cells obtained from the subject, wherein each of the one or more pairing sequences comprises a CDR3 sequence; and (b) identifying a tissue CDR3 sequence of a TCR chain of the TCR in the tissue sample, the other TCR chain naturally paired with the TCR chain being unknown, wherein the tissue CDR3 sequence matches the CDR3 sequence of at least one of the one or more pairing sequences of the one or more naturally paired TCRs, thereby identifying the at least one pairing sequence as the sequence of the naturally paired TCR in the tissue sample.

[0384]

[171] The method of paragraph

[170] , wherein the identification in (a) comprises sequencing the one or more naturally paired TCRs in the sample containing the plurality of peripheral T cells.

[0385]

[172] A method as described in paragraph

[171] , wherein the sequencing includes single-cell sequencing.

[0386]

[173] The method of paragraph

[172] , wherein the single-cell sequencing comprises dividing the plurality of peripheral T cells into a plurality of compartments, each compartment containing a single peripheral T cell from the plurality of peripheral T cells.

[0387]

[174] A method as described in any of paragraphs

[170] -

[173] , wherein the tissue sample is not a body fluid sample.

[0388]

[175] The method of any of paragraphs

[170] -

[174] , wherein the tissue sample is a solid tumor sample.

[0389]

[176] A method as described in any of paragraphs

[170] -

[175] , wherein the tissue sample is a fixed or frozen sample.

[0390]

[177] The method of any of paragraphs

[170] -

[176] , wherein the sample containing the plurality of peripheral T cells is a peripheral blood mononuclear cell (PBMC) sample.

[0391]

[178] The method of any of paragraphs

[170] -

[177] , further comprising obtaining a blood sample from the subject before (a).

[0392]

[179] The method of paragraph

[178] further comprises isolating peripheral blood mononuclear cells from the blood sample before (a).

[0393]

[180] The method of any of paragraphs

[170] -

[179] , wherein the tissue sample comprises tumor-infiltrating T cells.

[0394]

[181] A method for identifying a target-reactive T cell receptor (TCR), comprising: (a) providing a cell comprising the TCR identified in any one of paragraphs

[170] -

[180] ; and (b) contacting the cell with a target antigen presented by an antigen presenting cell (APC), wherein the cell binds to the target antigen presented by the APC through the TCR, thereby identifying the TCR as the target-reactive TCR.

[0395]

[182] The method of paragraph

[181] , wherein the target antigen is a tumor antigen.

[0396]

[183] ​​The method of paragraphs

[181] or

[182] , further comprising delivering a sequence encoding the target-reactive TCR into a host cell.

[0397]

[184] The method of paragraph

[183] ​​further comprises administering the host cell to the subject.

[0398]

[185] The method of paragraph

[183] ​​or

[184] , wherein the host cell is a T cell.

[0399]

[186] The method of paragraph

[185] , wherein the T cells are autologous T cells.

[0400]

[187] The method of paragraph

[185] , wherein the T cells are allogeneic T cells.

[0401]

[188] A method as described in any of paragraphs

[181] -

[187] , wherein the cell is a reporter cell line, which comprises a reporter gene expressed when the cell binds to the target antigen presented by the APC.

Claims

1. A method for producing a plurality of nucleic acid molecules encoding a plurality of different native T cell receptor (TCR) alpha chains or portions thereof in the same compartment, the method comprising: (a) providing a first plurality of nucleic acid molecules, each nucleic acid molecule in the first plurality of nucleic acid molecules comprising a sequence encoding a CDR3α of a different TCRα chain and a unique linker sequence; (b) providing a second plurality of nucleic acid molecules, each nucleic acid molecule of the second plurality comprising a sequence derived from a TCRαV (TRAV) gene and a unique anti-ligation sequence, wherein the second plurality of nucleic acid molecules comprises at least two different sequences derived from at least two different TRAV genes; and (c) producing a third plurality of nucleic acid molecules, each of the third plurality of nucleic acid molecules comprising a sequence from one of the sequences encoding CDR3α from the first plurality of nucleic acid molecules from (a) and a sequence from one of at least two different TRAV genes from the second plurality of nucleic acid molecules from (b), wherein the production comprises contacting the first plurality of nucleic acid molecules of (a) with the second plurality of nucleic acid molecules of (b) in the same compartment and specifically ligating a unique linking sequence of a nucleic acid molecule in the first plurality of nucleic acid molecules of (a) with a unique anti-linking sequence of a nucleic acid molecule in the second plurality of nucleic acid molecules of (b) comprising a sequence from a single given TRAV gene from the at least two different TRAV genes, wherein the unique anti-linking sequence is complementary to the unique linking sequence, thereby producing a plurality of said nucleic acid molecules encoding said plurality of different TCRα chains or portions thereof, and wherein the single given TRAV gene is homologous to the CDR3α to which it is linked.

2. The method of claim 1, wherein the sequence from the single given TRAV gene and the sequence encoding the CDR3α specifically linked thereto in (c) are from the same TCRα chain expressed in T cells from a sample from a subject.

3. The method of claim 1 or 2, wherein each nucleic acid molecule in the first plurality of nucleic acid molecules further comprises a J region of a TCR chain, and wherein the J region and the CDR3α are from the same TCRα chain.

4. The method of claim 1 or 2, wherein the at least two TRAV genes are human TRAV genes or mouse TRAV genes.

5. The method of claim 1 or 2, wherein the at least two TRAV genes are selected from human TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV7, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8-6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV14-2, TRAV15-3, TRAV16-4, TRAV17-5, TRAV18-6, TRAV19-1, TRAV19-2, TRAV20-1, TRAV21-2, TRAV22-3, TRAV23-1, TRAV24-2, TRAV25-3, TRAV26-4, TRAV27-5, TRAV28-6, TRAV29-1, TRAV29-2, TRAV30-1, TRAV31-2, TRAV32-3, TRAV33-1, TRAV34-2, TRAV35-3, TRAV36-4, TRAV37-5, TRAV38-6, TRAV39-1, TRAV31-2, TRAV30-3, TRAV31-4, TRAV37-6, TRAV38-1, TRAV39-2, TRAV30-1, TRAV31-2, TRAV31-3, TRAV33-1, TRAV34-2, TRAV35-3, TRAV36-4, TRAV37-5 The group consisting of TRAV13-2, TRAV14, TRAV16, TRAV17, TRAV18, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23, TRAV24, TRAV25, TRAV26-1, TRAV26-2, TRAV27, TRAV29, TRAV30, TRAV34, TRAV35, TRAV36, TRAV38-1, TRAV38-2, TRAV39, TRAV40 and TRAV41.

6. The method of claim 1, wherein the unique junction sequence or the unique anti-junction sequence encodes a portion of the TRAV gene adjacent to a sequence encoding CDR3α in a rearranged gene.

7. The method of claim 1 or 2, wherein each of the at least two sequences derived from the at least two different TRAV genes comprises a sequence encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and / or FR3.

8. The method of claim 2, wherein the subject has cancer.

9. The method of claim 1 or 2, wherein each nucleic acid molecule of the first plurality of nucleic acid molecules of (a) further comprises an additional sequence encoding CDR3β of a TCRβ chain.

10. The method of claim 9, wherein each nucleic acid molecule of the first plurality of nucleic acid molecules of (a) comprises an additional J region of the TCRβ chain, and wherein the additional J region and the CDR3β are from the same TCRβ chain.

11. The method of claim 9, wherein the sequence encoding the CDR3α and the additional sequence encoding the CDR3β are separated by at most 100 nucleotides.

12. The method of claim 9, wherein the TCR alpha chain and the TCR beta chain are a cognate pair of TCR chains.

13. The method of claim 1, wherein each unique joining sequence or each unique anti-joining sequence encodes the same amino acid sequence that is conserved in the TRAV gene adjacent to the sequence encoding the CDR3α in the rearranged gene.

14. The method of claim 1 or 2, wherein the nucleic acid molecules in the first plurality of nucleic acid molecules of (a) and the nucleic acid molecules in the second plurality of nucleic acid molecules of (b) encode functional TCR chains or portions thereof.

15. The method of claim 1 or 2, wherein the anti-ligation sequence is complementary to the ligation sequence of the nucleic acid molecules in the first plurality of nucleic acid molecules of (a).

16. The method of claim 13, wherein each unique junction sequence has a codon-diverse nucleic acid sequence.

17. The method of claim 2, wherein the sequence from the single given TRAV gene and the sequence encoding the CDR3α specifically linked thereto in (c) are from the same TCRα chain expressed in tumor-infiltrating T cells in the sample from the subject.

18. The method of claim 1 or 2, wherein ligation comprises hybridizing the ligation sequences of the nucleic acid molecules in the first plurality of nucleic acid molecules of (a) with the anti-ligation sequences of the nucleic acid molecules in the second plurality of nucleic acid molecules of (b).

19. The method of claim 18, further comprising (i) using the nucleic acid molecules in the first plurality of nucleic acid molecules of (a) as templates to extend the free 3' ends of the nucleic acid molecules in the second plurality of nucleic acid molecules, and / or (ii) using the nucleic acid molecules in the second plurality of nucleic acid molecules of (b) as templates to extend the free 3' ends of the nucleic acid molecules in the first plurality of nucleic acid molecules of (a) to produce nucleic acid molecules in the third plurality of nucleic acid molecules.

20. The method of claim 19, further comprising ligating the nucleic acid molecules of the first plurality of nucleic acid molecules of (a) to the nucleic acid molecules of the second plurality of nucleic acid molecules of (b).

21. The method of claim 20, further comprising contacting the nucleic acid molecules of the third plurality of nucleic acid molecules with a restriction enzyme to generate sticky ends.

22. The method of claim 21, further comprising contacting the nucleic acid molecules of the third plurality of nucleic acid molecules with additional nucleic acid molecules.

23. The method of claim 22, wherein the additional nucleic acid molecule encodes a constant region of a TCR chain or a portion thereof.

24. The method of claim 23, further comprising ligating the nucleic acid molecules of the third plurality of nucleic acid molecules to the additional nucleic acid molecules.

25. The method of claim 1 or 2, further comprising, before (a), sequencing a sample from a subject to obtain the sequence of a cognate pair of TCRs expressed in the subject.

26. The method of claim 1 or 2, wherein at least five different nucleic acid molecules encoding at least five different TCR alpha chains or portions thereof are produced in the same compartment.

27. The method of claim 1 or 2, wherein at least ten different nucleic acid molecules encoding at least ten different TCR alpha chains or portions thereof are produced in the same compartment.

28. The method of claim 1 or 2, wherein the same compartment is a well, a tube, or a droplet.

29. The method of claim 1 or 2, wherein the ligation sequence comprises a unique barcode.

30. The method of claim 29, wherein the unique barcode is a primer binding site.

31. The method of claim 30, further comprising amplifying the nucleic acid molecules in the third plurality of nucleic acid molecules using primers that target the unique barcodes.

32. The method of claim 1 or 2, further comprising expressing in a cell a sequence of at least one nucleic acid molecule among the plurality of nucleic acid molecules encoding the plurality of different TCR alpha chains or portions thereof produced in (c).

33. A composition comprising (a) a plurality of nucleic acid molecules, wherein each nucleic acid molecule in the plurality comprises (i) a unique anti-junctional sequence, (ii) a sequence derived from a T cell receptor (TCR) V gene and (iii) does not comprise a CDR3 sequence, wherein a first nucleic acid molecule in the plurality comprises a first anti-junctional sequence and a second nucleic acid molecule in the plurality comprises a second anti-junctional sequence, wherein the first anti-junctional sequence is different from the second anti-junctional sequence, wherein the first anti-junctional sequence and the second anti-junctional sequence encode the same amino acid sequence that is conserved in a TRAV gene adjacent to the sequence encoding the CDR3 in a rearranged gene, and wherein the sequences derived from the TCR V gene of the first nucleic acid molecule and the second nucleic acid molecule are derived from different TCR V genes; and (b) a first plurality of nucleic acid molecules, wherein each of the first plurality of nucleic acid molecules comprises a sequence encoding a different CDR3 of a different TCR chain, wherein each of the first plurality of nucleic acid molecules comprises a unique linking sequence, wherein the unique linking sequence is capable of specifically linking to a unique anti-linking sequence of a nucleic acid molecule comprising a sequence derived from a single given TCR V gene among the plurality of nucleic acid molecules of (a), wherein the unique anti-linking sequence is complementary to the unique linking sequence.

34. The composition of claim 33, wherein the composition is a liquid composition.

35. The composition of claim 33 or 34, wherein the plurality of nucleic acid molecules of (a) and the first plurality of nucleic acid molecules of (b) are in the same compartment.

36. A composition as described in claim 33 or 34, wherein the sequence derived from the TCR V gene comprises at least ten nucleotides of the TCR V gene.

37. A composition as described in claim 33 or 34, wherein the TCR V gene is a TRAV gene, a TRBV gene, a TRGV gene or a TRDV gene.

38. compositions as claimed in claim 33 or 34, wherein the sequence derived from the TCR V gene comprises the sequence of coding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and / or FR3.

39. A composition as described in claim 33 or 34, wherein each of the first plurality of nucleic acid molecules of (b) further comprises a J region of the TCR chain.

40. The composition of claim 33 or 34, wherein each of the first plurality of nucleic acid molecules of (b) further comprises additional sequences encoding additional CDR3s of additional TCR chains.

41. A composition as described in claim 40, wherein each of the first plurality of nucleic acid molecules of (b) further comprises an additional J region of the additional TCR chain.

42. The composition of claim 40, wherein the sequence encoding the CDR3 and the additional sequence encoding the CDR3 are separated by at most 100 nucleotides.

43. The composition of claim 40, wherein the TCR chain and the additional TCR chain are a cognate pair of TCR chains.

44. The composition of claim 33 or 34, wherein the first anti-ligation sequence or the second anti-ligation sequence is codon-diversified.

45. The composition of claim 33, wherein the unique linking sequence or the unique anti-linking sequence is greater than or equal to 15 nucleotides in length and less than or equal to 300 nucleotides in length.

46. ​​A composition as described in claim 33 or 34, wherein each of the multiple nucleic acid molecules of (a) does not contain a sequence encoding a constant domain of a TCR chain.

47. A composition as described in claim 33 or 34, wherein the unique junction sequence hybridizes to the unique anti-junction sequence of a nucleic acid molecule comprising a sequence derived from a single given TCR V gene among the multiple nucleic acid molecules of (a).

48. The composition of claim 33 or 34, wherein each nucleic acid molecule in the first plurality of nucleic acid molecules of (b) comprises a unique barcode.

49. The composition of claim 48, wherein the unique barcode is a primer binding site.

50. The composition of claim 33 or 34, wherein the unique junction sequence of each nucleic acid molecule of the first plurality of nucleic acid molecules of (b) comprises a unique barcode.

51. The composition of claim 50, wherein the unique barcode is a primer binding site.

52. A method for producing a plurality of nucleic acid molecules encoding a plurality of different native TCR chains or homologous pairs of TCR chains in the same compartment, comprising: (a) providing a first plurality of nucleic acid molecules, wherein each nucleic acid molecule of the first plurality of nucleic acid molecules comprises a sequence encoding a first CDR3 of a first T cell receptor (TCR) chain and a second CDR3 of a second TCR chain and a unique joining sequence, wherein the first CDR3 and the second CDR3 are from a cognate pair of TCR chains, and wherein each nucleic acid molecule encodes a first CDR3 and a second CDR3 from a different cognate pair of TCR chains; (b) providing a second plurality of nucleic acid molecules, wherein each nucleic acid molecule of the second plurality of nucleic acid molecules comprises a sequence derived from a different TCR V gene and a unique anti-ligation sequence; and (c) contacting the first plurality of nucleic acid molecules of (a) with the second plurality of nucleic acid molecules of (b), wherein the unique joining sequence of the nucleic acid molecules in the first plurality of nucleic acid molecules of (a) is specifically linked to the unique anti-joining sequence of the nucleic acid molecules in the second plurality of nucleic acid molecules of (b) to form a linear nucleic acid molecule comprising the sequences encoding the first CDR3 and the second CDR3 and the sequence derived from a single given TCR V gene, wherein the sequences encoding the first CDR3 and the second CDR3 and the single given TCR V gene are derived from a homologous pair of TCR chains, and wherein the unique anti-joining sequence is complementary to the unique joining sequence.

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