CN117015374A

CN117015374A - Ionizable cationic lipids and lipid nanoparticles and methods of synthesis and use thereof

Info

Publication number: CN117015374A
Application number: CN202180081874.2A
Authority: CN
Inventors: M·阿里; A·W·波斯奇; D·C·德拉蒙德; W·库尔曼; U·尼耳森
Original assignee: Tidal Therapy Co
Current assignee: Tidal Therapy Co
Priority date: 2020-12-04
Filing date: 2021-12-03
Publication date: 2023-11-07

Abstract

Ionizable cationic lipids and lipid nanoparticles for delivering nucleic acids to cells (e.g., immune cells) and methods of making and using such lipids and targeted lipid nanoparticles are provided.

Description

Ionizable cationic lipids and lipid nanoparticles and methods of synthesis and use thereof

Cross Reference to Related Applications

The present application claims priority and benefit from the following provisional applications: U.S. provisional application No. 63/121,801, filed 12/4/2020; U.S. provisional application Ser. No. 63/166,205, filed on 3/25 of 2021; U.S. provisional application No. 63/169,296, filed on 1/4/2021; U.S. provisional application No. 63/169,395, filed on 1/4/2021; and U.S. provisional application No. 63/172,024 filed on 7, 4, 2021, the entire disclosures of which are incorporated herein by reference in their entireties.

Submission of ASCII text file sequence Listing

The contents of the following submitted ASCII text files are incorporated herein by reference in their entirety: a sequence listing in Computer Readable Form (CRF) (file name: 1839520340 seqlist. Txt, date of record: 2021, 12, 3, size: 205,763 bytes).

Technical Field

The present invention provides ionizable cationic lipids and lipid nanoparticles for delivering nucleic acids to immune cells and methods of making and using such lipids and targeted lipid nanoparticles.

Background

In recent years, a number of therapeutic approaches have been developed that involve the delivery of one or more nucleic acids to a subject. Therapeutic approaches include, for example, gene therapy, in which a gene of interest in the form of deoxyribonucleic acid (DNA) is introduced into a cell, and then expressed to produce a gene product (e.g., a protein) for use in treating a disorder caused by or associated with a deficiency or deficiency of the gene product. In this method, a gene is transcribed into messenger ribonucleic acid (mRNA) and the mRNA is then translated to produce a gene product. In another approach, mRNA can be delivered to the cell instead of the gene of interest. The resulting expression products can ameliorate a defect or deficiency of a particular protein in a subject (e.g., a protein defect that occurs in some form of cystic fibrosis or lysosomal storage disorder), or can be used to modulate cellular functions, such as reprogramming immune cells to initiate or otherwise modulate an immune response in a subject (e.g., as a therapeutic for treating cancer or as a prophylactic vaccine for preventing or minimizing risk or severity of microbial or viral infection).

However, delivery of mRNA to cells for translation within the cell is challenging due to a variety of factors, such as nuclease degradation of the mRNA before entry into the cell and after introduction into the cell but prior to translation.

The RNA can be delivered to the subject using a different delivery vehicle, for example based on a cationic polymer or lipid that forms nanoparticles with the RNA. Nanoparticles are intended to protect RNA from degradation, enable delivery of RNA to a target site, and promote cellular uptake and processing by target cells. For delivery efficacy, parameters like particle size, charge or grafting to molecular moieties like polyethylene glycol (PEG) or ligands are also contributing in addition to molecular composition. Grafting with PEG is believed to reduce serum interactions, increase serum stability, and increase circulation time, which may be helpful for certain targeting approaches.

Compared to the DNA delivery techniques used in certain gene therapies, mRNA-based gene therapies have many advantageous features, such as ease of handling, rapid and transient expression, and adaptive switchability without mutagenesis.

However, in view of the relative instability of RNA and low cell permeability, delivering therapeutic RNA to cells is difficult. Thus, there is a need to develop methods and compositions for facilitating the delivery of RNA, such as mRNA, to cells.

Disclosure of Invention

The present invention provides ionizable cationic lipids, lipid-immune cell targeting group conjugates, and lipid nanoparticle compositions comprising such ionizable cationic lipids and/or lipid-immune cell (e.g., T cell) targeting group conjugates, medical kits containing such lipids and/or conjugates, and methods of making and using such lipids and conjugates.

The lipid nanoparticle compositions provided herein may further comprise a nucleic acid, such as RNA, e.g., messenger RNA or mRNA. The lipid nanoparticle composition can be used to deliver mRNA to cells (e.g., immune cells, such as T cells) of a subject. Messenger RNA-based gene therapy requires efficient delivery of mRNA to circulating cells (e.g., immune cells, such as T cells or NK cells) in plasma or to cells in a given tissue. Major challenges associated with efficient mRNA delivery to achieve robust protein expression levels include: (a) The ability to protect mRNA payloads from ubiquitous serum nucleases when administered to a subject; (b) The ability to specifically target mRNA delivery to a target cell (e.g., T cell) population, thereby maximizing protein expression therein; and (c) the ability to deliver mRNA payloads to the cytosolic compartment of cells (e.g., T cells) to be translated into proteins within the cytoplasm.

The present invention provides ionizable cationic lipids for use in the production of lipid nanoparticle compositions that facilitate delivery of a payload (e.g., a nucleic acid, such as DNA or RNA, such as mRNA) disposed therein to a cell, e.g., a mammalian cell, e.g., an immune cell. The lipids are designed to be able to deliver nucleic acids (e.g., mRNA) intracellularly to the cytoplasmic compartments of the target cell type and degrade rapidly into non-toxic components. These complex functions are achieved by interactions between the chemical and geometric shapes of the ionizable lipid head groups, the hydrophobic "acyl tail" groups, and the linkers that connect the head groups and acyl tail groups in the ionizable cationic lipid.

In one aspect, the present invention provides a compound represented by formula I:

or a salt thereof, wherein the variables are as defined herein.

In another aspect, the present invention provides a compound represented by formula II:

or a salt thereof, wherein the variables are as defined herein.

Provided herein, in part, is a compound selected from the group consisting of:

or a salt thereof.

In certain embodiments, the compound is a compound of formula III:

or a salt thereof, wherein the variables are as defined herein.

Also provided herein is a compound of the formula:

or a salt thereof.

Also provided herein is a compound of the formula:

or a salt thereof.

Also provided herein is a compound of the formula:

or a salt thereof.

Also provided herein is a compound of the formula:

or a salt thereof.

Also provided herein is a compound of the formula:

or a salt thereof.

Also provided herein is a compound of the formula:

or a salt thereof.

Also provided herein is a compound of the formula:

or a salt thereof.

Also provided herein is a compound of the formula:

or a salt thereof.

Also provided herein is a Lipid Nanoparticle (LNP) comprising a lipid blend comprising an ionizable cationic lipid and/or a lipid-immune cell targeting group conjugate (e.g., a lipid-T cell targeting group conjugate) provided herein.

In another aspect, provided herein is a method of delivering a nucleic acid to an immune cell (e.g., a T cell), the method comprising exposing the immune cell to an LNP containing the nucleic acid described herein under conditions that allow the nucleic acid to enter the immune cell.

In another aspect, provided herein is a method of delivering a nucleic acid to an immune cell (e.g., a T cell) of a subject in need thereof, the method comprising administering to the subject a composition comprising an LNP containing a nucleic acid as described herein, thereby delivering the nucleic acid to the immune cell.

In another aspect, provided herein is a method of targeting delivery of a nucleic acid (e.g., mRNA) to an immune cell (e.g., T cell) of a subject, the method comprising administering to the subject an LNP containing the nucleic acid described herein to facilitate targeted delivery of the nucleic acid to the immune cell.

In one aspect, provided herein are Lipid Nanoparticles (LNPs) for targeted delivery of nucleic acids to immune cells comprising a lipid blend. In some embodiments, the lipid blend comprises a lipid-immune cell targeting group conjugate comprising a compound of formula IV: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the lipid blend comprises an ionizable cationic lipid. In some embodiments, the ionizable cationic lipid comprises

In some embodiments, the LNP comprises a nucleic acid disposed therein.

In some embodiments, the immune cell targeting group comprises an antibody that binds a T cell antigen. In some embodiments, the T cell antigen is CD3, CD4, CD7, or CD8, or a combination thereof (e.g., both CD3 and CD8, both CD4 and CD8, or both CD7 and CD 8). In some embodiments, the immune cell targeting group comprises an antibody that binds a Natural Killer (NK) cell antigen. In some embodiments, the NK cell antigen is CD7, CD8, or CD56, or a combination thereof (e.g., both CD7 and CD 8). In some embodiments, the antibody is a human or humanized antibody.

In some embodiments, the immune cell targeting group is covalently coupled to the lipids in the lipid blend via a linker comprising polyethylene glycol (PEG). In some embodiments, the lipid covalently coupled to the immune cell targeting group via a PEG-containing linker is distearoyl glycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycerol-phosphate glycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (dpp), or ceramide. In some embodiments, the PEG is PEG 2000.

In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.002-0.2 mole percent. In some embodiments, the lipid blend comprises one or more of a structural lipid (e.g., a sterol), a neutral phospholipid, and a free PEG-lipid. In some embodiments, the ionizable cationic lipid is present in the lipid blend in a range of 40-60 mole percent. In some embodiments, the sterols are present in the lipid blend in the range of 30-50 mole percent. In some embodiments, the sterols are present in the lipid blend in the range of 20-70 mole percent. In some embodiments, the sterol is cholesterol.

In some embodiments, the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM). In some embodiments, the neutral phospholipid is present in the lipid blend in a range of 1-10 mole percent.

In some embodiments, the free PEG-lipid is selected from PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), N- (methylpolyoxyoxycarbonyl) -1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1, 2-dimyristoyl-rac-glycero-3-methylpolyethylene oxide (PEG-DMG), 1, 2-dimyristoyl-rac-glycero-3-methylpolyethylene oxide (PEG-dpp), 1, 2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG), 1, 2-distearoyl-rac-glycero-3-methylpolyethylene oxide (PEG-DSG), N-palmitoyl-sphingosine-1- { succinyl [ methoxy (polyethylene glycol) ] (PEG-ceramide), and DSPE-PEG-cysteine, or derivatives thereof. In some embodiments, the free PEG-lipid comprises a diacyl phosphatidylethanolamine comprising a dipalmitoyl (C16) chain or a distearoyl (C18) chain. In some embodiments, the free PEG-lipid is a mixture of two or more unique free PEG-lipids. In some embodiments, the free PEG-lipid is present in the lipid blend in a range of 1-4 mole percent (e.g., about 1-2 mole percent, or about 2-4 mole percent, or about 1.5 mole percent). In some embodiments, the free PEG-lipid comprises the same or different lipid as the lipid in the lipid-immune cell targeting group conjugate.

In some embodiments, the LNP has an average diameter in the range of 50-200 nm. In some embodiments, the LNP has an average diameter of about 100 nm. In some embodiments, the LNP has a polydispersity index in the range of from 0.05 to 1. In some embodiments, the LNP has a zeta potential of from about-10 mV to about +30mV at pH 5.

In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA, tRNA, siRNA or microrna. In some embodiments, the mRNA encodes a receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen, an enzyme, or a vaccine. In some embodiments, the mRNA encodes a polypeptide capable of modulating an immune response in the immune cell. In some embodiments, the mRNA encodes a polypeptide that is capable of reprogramming the immune cell. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or Chimeric Antigen Receptor (CAR). In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 24. In some embodiments, the mRNA encoding the CAR comprises the polynucleotide sequence of SEQ ID NO. 25.

In some embodiments, the immune cell targeting group comprises an antibody, and the antibody is a Fab or immunoglobulin single variable domain, such as a nanobody. In some embodiments, the immune cell targeting group comprises a Fab, F (ab ') 2, fab' -SH, fv, or scFv fragment. In some embodiments, the immune cell targeting group comprises a Fab engineered to knock out one or more native interchain disulfide bonds. For example, in some embodiments, the Fab comprises a heavy chain fragment comprising a C233S substitution, numbered according to Kabat; and/or light chain fragments containing a C214S substitution, numbered according to Kabat. In some embodiments, the immune cell targeting group comprises a Fab engineered to introduce one or more embedded interchain disulfide bonds. For example, in some embodiments, the Fab antibody comprises a heavy chain fragment comprising an F174C substitution, numbered according to Kabat; and/or light chain fragments containing S176C substitution, numbered according to Kabat. In some embodiments, the immune cell targeting group comprises a Fab engineered to knock out one or more native interchain disulfide bonds and introduce one or more embedded interchain disulfide bonds. In some embodiments, the immune cell targeting group comprises a Fab containing a cysteine at the C-terminus of the heavy or light chain fragment. In some embodiments, the Fab further comprises one or more amino acids between the heavy chain fragment of the Fab and the C-terminal cysteine. In some embodiments, the Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and wherein the immune cell targeting group further comprises a single chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain by an amino acid linker. In some embodiments, the Fab antibody is a DS Fab (Fab with wild-type (natural) interchain disulfide bond), a NoDS Fab (Fab with knocked-out natural disulfide bond, such as a Fab with C233S substitution on the heavy chain and/or C214S substitution on the light chain, according to Kabat numbering), a bDS Fab (Fab without natural disulfide bond and with non-natural interchain embedded disulfide bond introduced, such as a Fab with F174C and C233S on the heavy chain and/or S176C and C214S substitution on the light chain, according to Kabat numbering), or a bDS Fab-ScFv (bDS Fab linked to by a linker (such as (G4S) x) and ScFv), as shown in fig. 47.

In some embodiments, the immune cell targeting group comprises an immunoglobulin single variable domain, such as a nanobody. In some embodiments, the immunoglobulin single variable domain comprises a cysteine at the C-terminus. In some embodiments, the nanobody further comprises a spacer comprised in the V _HH One or more amino acids between the domain and the C-terminal cysteine. In some embodiments, the immune cell targeting group comprises two or more V _HH A domain. In some embodiments, the two or more V _HH The domains are linked by amino acid linkers. In some embodiments, the immune cell targeting group comprises a first V linked to an antibody CH1 domain _HH Domain and second V linked to antibody light chain constant domain _HH A domain, and wherein the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds. In some embodiments, the immune cell targeting group comprises V linked to an antibody CH1 domain _HH A domain, and wherein the antibody CH1 domain is linked to an antibody light chain constant domain by one or more disulfide bonds. In some embodiments, the CH1 domain comprises F174C and C233S substitutions, and/or the light chain constant domain comprises S176C and C214S substitutions, numbered according to Kabat. In some embodiments, the antibody is an ScFv, V _HH (Nb)、2xV _HH 、V _HH -CH 1/empty Vk or V _HH 1-CH1/V _HH -2-Nb bDS, as shown in fig. 47.

In some embodiments, the immune cell targeting group comprises a Fab comprising a heavy chain fragment comprising the amino acid sequence of SEQ ID No. 1 and a light chain fragment comprising the amino acid sequence of SEQ ID No. 2 or 3. In some embodiments, the immune cell targeting group comprises a Fab comprising a heavy chain fragment comprising the amino acid sequence of SEQ ID No. 6 and a light chain fragment comprising the amino acid sequence of SEQ ID No. 7. In some embodiments, the antibody is an antibody described in the examples.

In some embodiments, the immune cell targeting group comprises a Fab comprising:

(a) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 2 or 3;

(b) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 4 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 5;

(c) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 7;

(d) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 8 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 9;

(e) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 10 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 11;

(f) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 12 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 13;

(g) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 14 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 15;

(h) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 16 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 17;

(i) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 18 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 19;

(j) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 20 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 21; or alternatively

(k) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 22 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 23.

In some embodiments, the immune cell targeting group comprises a Fab, F (ab ') 2, fab' -SH, fv, or scFv fragment. In some embodiments, the immune cell targeting group comprises a Fab engineered to knock out the natural interchain disulfide bond at the C-terminus. In some embodiments, the Fab comprises a heavy chain fragment comprising a C233S substitution and a light chain fragment comprising a C214S substitution, numbered according to Kabat. In some embodiments, the immune cell targeting group comprises a Fab having non-native interchain disulfide bonds (e.g., engineered embedded interchain disulfide bonds). In some embodiments, the Fab comprises an F174C substitution in the heavy chain fragment and an S176C substitution in the light chain fragment, numbered according to Kabat. In some embodiments, the immune cell targeting group comprises a Fab containing a cysteine at the C-terminus of the heavy or light chain fragment. In some embodiments, the Fab further comprises one or more amino acids between the heavy chain fragment of the Fab and the C-terminal cysteine.

In some embodiments, the immune cell targeting group comprises an immunoglobulin single variable domain. In some embodiments, the immunoglobulin single variable domain comprises a cysteine at the C-terminus. In some embodiments, the immunoglobulin single variable domain comprises a VHH domain, and further comprises a spacer comprising one or more amino acids between the VHH domain and the C-terminal cysteine. In some embodiments, the immune cell targeting group comprises two or more VHH domains. In some embodiments, the two or more V _HH The domains are linked by amino acid linkers. In some embodiments, the immune cell targeting group comprises a first V linked to an antibody CH1 domain _HH Domain and second V linked to antibody light chain constant domain _HH A domain. In some embodiments, the antibody CH1 domain and the antibodyThe light chain constant domains are linked by one or more disulfide bonds. In some embodiments, the immune cell targeting group comprises a VHH domain linked to an antibody CH1 domain. In some embodiments, the antibody CH1 domain is linked to an antibody light chain constant domain by one or more disulfide bonds. In some embodiments, the CH1 domain comprises F174C and C233S substitutions and the light chain constant domain comprises S176C and C214S substitutions, numbered according to Kabat.

In some embodiments, the immune cell targeting group comprises a Fab comprising: (a) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 2 or 3; or (b) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 7.

In another aspect, provided herein are methods of targeting delivery of a nucleic acid to an immune cell of a subject. In some embodiments, the method comprises contacting the immune cell with a Lipid Nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising a compound of the formula: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid.

In some embodiments, one aspect of the disclosure relates to LNP or pharmaceutical compositions containing the same, as disclosed herein, for use in a method of targeting delivery of a nucleic acid to immune cells of a subject. Such methods may be used to treat diseases or disorders as disclosed below. In some embodiments, the methods as disclosed herein may comprise contacting the immune cells of a subject with Lipid Nanoparticles (LNPs) in vitro or ex vivo. In some embodiments, the LNP is an LNP as described herein in the present disclosure.

In some embodiments, the LNP provides at least one of the following benefits:

(i) An increase in specificity of targeted delivery to the immune cells compared to a reference LNP;

(ii) An increase in half-life of the nucleic acid or polypeptide encoded by the nucleic acid in the immune cell compared to a reference LNP;

(iii) Increased transfection efficiency compared to reference LNP; and

(iv) Low levels of dye accessible mRNA (< 15%) and high RNA encapsulation efficiency, with at least 80% of the mRNA recovered in the final formulation relative to the total RNA used in the LNP batch preparation.

In some aspects, methods of expressing a polypeptide of interest in a targeted immune cell of a subject are provided. In some embodiments, the method comprises contacting the immune cell with a Lipid Nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid encoding the polypeptide. In some embodiments, one aspect of the disclosure relates to LNP or pharmaceutical compositions containing the same, as disclosed herein, for use in a method of expressing a polypeptide of interest in a targeted immune cell of a subject. Such methods may be used to treat diseases or disorders as disclosed below. In some embodiments, the methods as disclosed herein may comprise contacting the immune cells of a subject with Lipid Nanoparticles (LNPs) in vitro or ex vivo.

In some embodiments, the LNP provides at least one of the following benefits:

(i) Increased expression levels in the immune cells compared to a reference LNP;

(ii) Increased specificity of expression in the immune cells compared to a reference LNP;

(iii) An increase in half-life of the nucleic acid or polypeptide encoded by the nucleic acid in the immune cell compared to a reference LNP;

(iv) Increased transfection efficiency compared to reference LNP; and

(v) Low levels of dye accessible mRNA (< 15%) and high RNA encapsulation efficiency, with at least 80% of the mRNA recovered in the final formulation relative to the total RNA used in the LNP batch preparation.

In some aspects, methods of modulating cellular function of a target immune cell in a subject are provided. In some embodiments, the method comprises administering Lipid Nanoparticles (LNPs) to the subject. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid encoding a polypeptide for modulating cellular function of the immune cell. In some embodiments, one aspect of the disclosure relates to LNP or pharmaceutical compositions containing the same, as disclosed herein, for use in a method of modulating cellular function of a targeted immune cell in a subject. Such methods may be used to treat diseases or disorders as disclosed below. In some embodiments, the methods as disclosed herein may comprise contacting the immune cells of a subject with Lipid Nanoparticles (LNPs) in vitro or ex vivo.

In some embodiments, the LNP provides at least one of the following benefits:

(iv) Increased transfection efficiency compared to reference LNP;

(v) The LNP can be administered at a lower dose than the reference LNP to achieve the same biological effect in the immune cells; and

(vi) Low levels of dye accessible mRNA (< 15%) and high RNA encapsulation efficiency, with at least 80% of the mRNA recovered in the final formulation relative to the total RNA used in the LNP batch preparation.

In some embodiments, the modulation of cellular function comprises reprogramming the immune cells to initiate an immune response. In some embodiments, the modulation of cellular function comprises modulating antigen specificity of the immune cell.

In some aspects, methods of treating, ameliorating, or preventing a symptom of a disorder or disease in a subject in need thereof are provided. In some embodiments, the method comprises administering Lipid Nanoparticles (LNPs) to the subject to deliver nucleic acid to immune cells of the subject. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid.

In some embodiments, the nucleic acid modulates an immune response of the immune cell, thereby treating or ameliorating the symptom. In some embodiments, one aspect of the disclosure relates to LNP or pharmaceutical compositions containing the same, as disclosed herein, for use in a method of treating, ameliorating or preventing a symptom of a disorder or disease in a subject in need thereof. The disease or disorder may be as disclosed below. In some embodiments, the methods as disclosed herein may comprise contacting the immune cells of a subject with Lipid Nanoparticles (LNPs) in vitro or ex vivo.

In some embodiments, the LNP provides at least one of the following benefits:

(i) An increase in specificity of delivering the nucleic acid to the immune cell compared to a reference LNP;

(iii) Increased transfection efficiency compared to reference LNP;

(iv) The LNP can be administered at a lower dose than the reference LNP to achieve the same therapeutic efficacy;

(v) Increased levels of functional gain of immune cells compared to reference LNP; and

In some embodiments, the disorder is an immune disorder, an inflammatory disorder, or cancer. In some embodiments, the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine for treating or preventing a pathogen infection. In some embodiments, the Fab antibody comprises a heavy chain fragment comprising an F174C substitution, numbered according to Kabat; and/or light chain fragments containing S176C substitution, numbered according to Kabat.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the non-immune cells are transfected with the LNP. In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the unwanted immune cells that are not intended to be the target of the delivery are transfected by the LNP. In some embodiments, the half-life of a nucleic acid delivered to the immune cell by the LNP or a polypeptide encoded by the nucleic acid delivered to the LNP is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more longer than the half-life of a polypeptide encoded by a nucleic acid delivered to the immune cell by a reference LNP or the nucleic acid delivered by the reference LNP.

In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the immune cells intended to be targeted for said delivery are transfected by said LNP.

In some embodiments of the present invention, in some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 5% higher than the expression level of the nucleic acid delivered by a reference LNP in the same immune cell at least 10%, at least 10% >, at least 10%, at least 10% >, at least 10%, at least 10% >.

In one aspect, lipid Nanoparticles (LNPs) for delivering nucleic acids to NK cells of a subject are provided. The LNP comprises (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]; (c) sterols or other structural lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipid; and (f) the nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds CD 56.

In one aspect, lipid Nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. The LNP comprises (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]; (c) sterols or other structural lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipid; and (f) the nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds CD7 or CD8, and the free PEG lipid is DMG-PEG.

In one aspect, there is provided an immune cell for delivering a nucleic acid to a subjectLipid Nanoparticles (LNP). The LNP comprises (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [ lipid ]]- [ optional linker ]]- [ immune cell targeting group ]]The method comprises the steps of carrying out a first treatment on the surface of the (c) sterols or other structural lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipid; and (f) the nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody, and the antibody is a Fab or immunoglobulin single variable domain. In some embodiments, the Fab is engineered to knock out the natural interchain disulfide bond at the C-terminus. In some embodiments, the Fab has an embedded interchain disulfide linkage. In some embodiments, the antibody is an Immunoglobulin Single Variable (ISV) domain, and the ISV domain is ISV. In some embodiments, the free PEG lipid comprises PEG having a molecular weight of at least 2000 daltons. In some embodiments, the PEG has a molecular weight of about 3000 to 5000 daltons. In some embodiments, the Fab is an anti-CD 3 antibody and the free PEG lipid in the LNP comprises PEG having a molecular weight of about 2000 daltons. In some embodiments, the Fab is an anti-CD 4 antibody and the free PEG lipid in the LNP comprises PEG having a molecular weight of about 3000 to 3500 daltons.

In one aspect, lipid Nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. The LNP comprises (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]; (c) sterols or other structural lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipid; and (f) the nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds CD3 and an antibody that binds CD11a or CD 18.

In one aspect, lipid Nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. The LNP comprises (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]; (c) sterols or other structural lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipid; and (f) the nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds CD7 and an antibody that binds CD 8.

In one aspect, lipid Nanoparticles (LNPs) for delivering nucleic acids to two different types of immune cells of a subject are provided. The LNP comprises (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]; (c) sterols or other structural lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipid; and (f) the nucleic acid.

In some embodiments, the immune cell targeting moiety comprises a bispecific targeting moiety. In some embodiments, the bispecific targeting moiety binds to the two different types of immune cells. In some embodiments, the two different types of immune cells are cd4+ T cells and cd8+ T cells. In some embodiments, the bispecific targeting moiety is a bispecific antibody. In some embodiments, the bispecific antibody is a Fab-ScFv.

In one aspect, lipid Nanoparticles (LNPs) for delivering nucleic acids to both cd4+ and cd8+ T cells of a subject are provided. The LNP comprises (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]; (c) sterols or other structural lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipid; and (f) the nucleic acid. In some embodiments, the immune cell targeting group comprises a single antibody that binds to CD3 or CD 7.

Further provided is a Lipid Nanoparticle (LNP) for delivering a nucleic acid to an immune cell of a subject, wherein the LNP comprises: (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]; (c) sterols or other structural lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipid; and (f) the nucleic acid, wherein the immune cell targeting group comprises a Fab lacking native interchain disulfide bonds. In some embodiments, the Fab is engineered to replace one or both cysteines on the natural constant light chain and the natural constant heavy chain that form natural interchain disulfide bonds with non-cysteine amino acids, thereby removing the natural interchain disulfide bonds in the Fab.

An Immunoglobulin Single Variable Domain (ISVD) that binds to human CD8 is also provided. In some embodiments, the ISVD comprises three complementarity determining domains CDR1, CDR2, and CDR3, wherein

(a) The CDR1 comprises GSTFSDYG (SEQ ID NO: 100),

(b) The CDR2 comprises an IDWNGEHT (SEQ ID NO: 101), an

(c) The CDR3 includes AADALPYTVRKYNY (SEQ ID NO: 102).

In some embodiments, the ISVD is humanized.

In some embodiments, the ISVD comprises, consists of, or consists essentially of SEQ ID NO 77.

Also provided is a polypeptide comprising GSTFSDYG (SEQ ID NO: 100), IDWNGEHT (SEQ ID NO: 101) and AADALPYTVRKYNY (SEQ ID NO: 102).

In some embodiments, the polypeptide comprises ISVD as described herein.

In some embodiments, the polypeptide further comprises a second binding moiety, wherein the second binding moiety binds to CD8 or another different target. In some embodiments, the second binding moiety is also ISVD.

In some embodiments, the polypeptide further comprises a detectable label or therapeutic agent.

Also provided is a composition comprising an ISVD or a polypeptide as described herein.

Further provided is a pharmaceutical composition comprising an ISVD or a polypeptide as described herein and a pharmaceutically acceptable carrier.

Further provided is a method of treating a CD 8-associated disease or disorder in a subject, the method comprising administering to the subject a pharmaceutical composition as described herein.

In some embodiments, the disease is cancer. In some embodiments, the disorder is an immune disorder, an inflammatory disorder, or cancer.

In some embodiments, the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine for treating or preventing a pathogen infection. In some embodiments, the ionizable cationic lipid is

In some embodiments, the immune cell targeting group comprises an antibody that binds a T cell antigen. In some embodiments, the T cell antigen is CD3, CD8, or both CD3 and CD 8. 60. In some embodiments, the immune cell targeting group comprises an antibody that binds a Natural Killer (NK) cell antigen. In some embodiments, the NK cell antigen is CD56. In some embodiments, the antibody is a human or humanized antibody.

In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.002-0.2 mole percent. In some embodiments, the ionizable cationic lipid is present in the lipid blend in a range of 40-60 mole percent.

In some embodiments, the sterol is cholesterol. In some embodiments, the sterols are present in the lipid blend in the range of 30-50 mole percent. In some embodiments, the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM).

In some embodiments, the neutral phospholipid is present in the lipid blend in a range of 1-10 mole percent.

In some embodiments, the free PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, the PEG lipid may be PEG-dioleoyl glycerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-dppg), PEG-diiodoyl-glycerol-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoyl glycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycerol-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycerol-phosphoethanolamine (PEG-DOPE), 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide, or PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

In some embodiments, the free PEG-lipid comprises a diacyl phosphatidylethanolamine comprising a dipalmitoyl (C16) chain or a distearoyl (C18) chain. In some embodiments, the free PEG-lipid is present in the lipid blend in a range of 2-4 mole percent. In some embodiments, the free PEG-lipid comprises the same or different lipid as the lipid in the lipid-immune cell targeting group conjugate.

In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA, tRNA, siRNA or microrna. In some embodiments, the mRNA encodes a receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen, an enzyme, or a vaccine. In some embodiments, the mRNA encodes a polypeptide capable of modulating an immune response in the immune cell. In some embodiments, the mRNA encodes a polypeptide that is capable of reprogramming the immune cell. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or Chimeric Antigen Receptor (CAR).

In some embodiments, the immune cell targeting group comprises an antibody, and the antibody is a Fab or immunoglobulin single variable domain. In some embodiments, the immune cell targeting group comprises an antibody fragment selected from the group consisting of: fab, F (ab ') 2, fab' -SH, fv and scFv fragments. In some embodiments, the immune cell targeting group comprises a Fab containing one or more interchain disulfide bonds. In some embodiments, the Fab comprises a heavy chain fragment comprising F174C and C233S substitutions and a light chain fragment comprising S176C and C214S substitutions, numbered according to Kabat. In some embodiments, the immune cell targeting group comprises a Fab containing a cysteine at the C-terminus of the heavy or light chain fragment.

In some embodiments, the Fab further comprises one or more amino acids between the heavy chain fragment of the Fab and the C-terminal cysteine. In some embodiments, the Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain. In some embodiments, the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds. In some embodiments, the immune cell targeting group further comprises a single chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain via an amino acid linker.

In some embodiments, the immune cell targeting group comprises an immunoglobulin single variable domain. In some embodiments, the immunoglobulin single variable domain comprises a cysteine at the C-terminus. In some embodiments, the immunoglobulin single variable domain comprises a VHH domain, and further comprises a spacer comprising one or more amino acids between the VHH domain and the C-terminal cysteine. In some embodiments, the immune cell targeting group comprises two or more VHH domains. In some embodiments, the two or more VHH domains are linked by an amino acid linker. In some embodiments, the immune cell targeting group comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain. In some embodiments, the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds. In some embodiments, the immune cell targeting group comprises a VHH domain linked to an antibody CH1 domain. In some embodiments, the antibody CH1 domain is linked to an antibody light chain constant domain by one or more disulfide bonds. In some embodiments, the CH1 domain comprises F174C and C233S substitutions and the light chain constant domain comprises S176C and C214S substitutions, numbered according to Kabat.

(b) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 7.

In some embodiments, no more than 5% of the non-immune cells are transfected with the LNP. In some embodiments, the half-life of the nucleic acid delivered by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP is at least 10% longer than the half-life of the nucleic acid delivered by the reference LNP or the polypeptide encoded by the nucleic acid delivered by the reference LNP. In some embodiments, at least 10% of the immune cells are transfected with the LNP. In some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 10% higher than the expression level of the nucleic acid delivered by the reference LNP.

In some aspects, lipid Nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid. In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cell targeting group comprises an antibody that binds CD 56.

In some aspects, provided herein are Lipid Nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds CD7 or CD 8. In some embodiments, the free PEG lipid is DMG-PEG.

In some aspects, lipid Nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody. In some embodiments, the antibody is a Fab or immunoglobulin single variable domain.

In some embodiments, the Fab is engineered to knock out the natural interchain disulfide bond at the C-terminus. In some embodiments, the Fab comprises a heavy chain fragment comprising a C233S substitution and a light chain fragment comprising a C214S substitution. In some embodiments, the Fab comprises a non-natural interchain disulfide bond. In some embodiments, the Fab comprises an F174C substitution in the heavy chain fragment and an S176C substitution in the light chain fragment. In some embodiments, the antibody is an Immunoglobulin Single Variable (ISV) domain, and the ISV domain isISV. In some embodiments, the free PEG lipid comprises PEG having a molecular weight of at least 2000 daltons. In some embodiments, the PEG has a molecular weight of about 3000 to 5000 daltons. In some embodiments, the antibody is a Fab. In some embodiments, the Fab binds to CD3 and the free PEG lipid in the LNP comprises PEG having a molecular weight of about 2000 daltons. In some embodiments, the Fab is an anti-CD 4 antibody and the free PEG lipid in the LNP comprises PEG having a molecular weight of about 3000 to 3500 daltons.

In some embodiments, the immune cell targeting group comprises two or more VHH domains. In some embodiments, the two or more VHH domains are linked by an amino acid linker. In some embodiments, the immune cell targeting group comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain.

In some aspects, lipid Nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid.

In some embodiments, the LNP binds to CD3 and also binds to CD11a or CD18. In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate comprises an antibody that binds CD 3. In some embodiments, the second conjugate comprises an antibody that binds CD11a or CD18. In some embodiments, the LNP comprises a conjugate. In some embodiments, the conjugate comprises a bispecific antibody that binds both CD3 and CD11 a. In some embodiments, the conjugate comprises a bispecific antibody that binds both CD3 and CD18. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

In some aspects, lipid Nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid. In some embodiments, the LNP binds to CD7 and CD8 of the immune cell.

In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate comprises an antibody that binds CD7 and the second conjugate comprises an antibody that binds CD8. In some embodiments, the LNP comprises a conjugate. In some embodiments, the conjugate comprises a bispecific antibody that binds CD7 and CD8. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

In some aspects, lipid Nanoparticles (LNPs) for delivering nucleic acids to two different types of immune cells of a subject are provided. In some embodiments, the LNP comprises: the cationic lipid can be ionized. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid. In some embodiments, the LNP binds to a first antigen on the surface of a first type of immune cell and also binds to a second antigen on the surface of a second type of immune cell. In some embodiments, the two different types of immune cells are cd4+ T cells and cd8+ T cells. In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate comprises a first antibody that binds to the first antigen of the first type of immune cell and the second conjugate comprises a second antibody that binds to the second antigen of the second type of immune cell. In some embodiments, the LNP comprises a conjugate. In some embodiments, the conjugate comprises a bispecific antibody. In some embodiments, the bispecific antibody binds to both the first antigen on the first type of immune cell and the second antigen on the second type of immune cell. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

In some aspects, lipid Nanoparticles (LNPs) for delivering nucleic acids to both cd4+ and cd8+ T cells of a subject are provided. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid. In some embodiments, the immune cell targeting group comprises a single antibody that binds to CD3 or CD 7.

In some aspects, lipid Nanoparticles (LNPs) are provided for delivering nucleic acids to both T cells and NK cells of a subject. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid. In some embodiments, the immune cell targeting group binds to CD7, CD8, or both CD7 and CD 8.

In some aspects, lipid Nanoparticles (LNPs) are provided for delivering nucleic acids to both T cells and NK cells of a subject. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the structure: [ lipid ] - [ optional linker ] - [ immune cell targeting group ]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises the nucleic acid. In some embodiments, the immune cell targeting group is identical to both (i) CD3 and CD 56; (ii) both CD8 and CD 56; or (iii) both CD7 and CD 56.

In some embodiments, the immune cell targeting group is covalently coupled to the lipids in the lipid blend via a linker comprising polyethylene glycol (PEG). In some embodiments, the lipid covalently coupled to the immune cell targeting group via a PEG-containing linker is distearoyl glycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycerol-phosphate glycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (dpp), or ceramide. In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.002-0.2 mole percent. In some embodiments, the lipid blend further comprises one or more of a structural lipid (e.g., a sterol), a neutral phospholipid, and a free PEG-lipid.

In some embodiments, the ionizable cationic lipid is present in the lipid blend in a range of 40-60 mole percent.

In some embodiments, the sterols are present in the lipid blend in the range of 30-50 mole percent. In some embodiments, the sterol is cholesterol.

In some embodiments, the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the neutral phospholipid is present in the lipid blend in a range of 1-10 mole percent.

In some embodiments, the free PEG-lipid is selected from PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), N- (methylpolyoxyoxycarbonyl) -1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1, 2-dimyristoyl-rac-glycero-3-methylpolyethylene oxide (PEG-DMG), 1, 2-dimyristoyl-rac-glycero-3-methylpolyethylene oxide (PEG-dpp), 1, 2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG), 1, 2-distearoyl-rac-glycero-3-methylpolyethylene oxide (PEG-DSG), N-palmitoyl-sphingosine-1- { succinyl [ methoxy (polyethylene glycol) ] (PEG-ceramide), and DSPE-PEG-cysteine, or derivatives thereof. In some embodiments, the free PEG-lipid comprises a diacyl phosphatidylethanolamine comprising a dipalmitoyl (C16) chain or a distearoyl (C18) chain. In some embodiments, the free PEG-lipid is present in the lipid blend in a range of 1-2 mole percent. In some embodiments, the free PEG-lipid comprises the same or different lipid as the lipid in the lipid-immune cell targeting group conjugate.

In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA. In some embodiments, the mRNA encodes a receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen, an enzyme, or a vaccine. In some embodiments, the mRNA encodes a polypeptide capable of modulating an immune response in the immune cell. In some embodiments, the mRNA encodes a polypeptide that is capable of reprogramming the immune cell. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or Chimeric Antigen Receptor (CAR).

In some embodiments, the immune cell targeting group comprises a Fab lacking native interchain disulfide bonds. In some embodiments, the Fab is engineered to replace one or both cysteines on the natural constant light chain and the natural constant heavy chain that form natural interchain disulfide bonds with non-cysteine amino acids, thereby removing the natural interchain disulfide bonds in the Fab.

In some aspects, methods of targeting delivery of a nucleic acid to an immune cell of a subject are provided. In some embodiments, the method comprises contacting the immune cell with a Lipid Nanoparticle (LNP) provided herein. In some embodiments, the methods are for targeting NK cells. In some embodiments, the immune cell targeting group binds to CD 56. In some embodiments, the method is for targeting both T cells and NK cells simultaneously. In some embodiments, the immune cell targeting group binds to CD7, CD8, or both CD7 and CD 8. In some embodiments, the methods are used to target both cd4+ and cd8+ T cells simultaneously. In some embodiments, the immune cell targeting group comprises a polypeptide that binds to CD3 or CD 7.

In some aspects, methods of expressing a polypeptide of interest in a targeted immune cell of a subject are provided. In some embodiments, the method comprises contacting the immune cell with a Lipid Nanoparticle (LNP) provided herein.

In some aspects, methods of modulating cellular function of a target immune cell in a subject are provided. In some embodiments, the method comprises administering to the subject a Lipid Nanoparticle (LNP) provided herein.

In some aspects, methods of treating, ameliorating, or preventing a symptom of a disorder or disease in a subject in need thereof are provided. In some embodiments, the method comprises administering to the subject a Lipid Nanoparticle (LNP) provided herein.

In some aspects, immunoglobulin Single Variable Domains (ISVD) that bind to human CD8 are provided. In some embodiments, the ISVD comprises three complementarity determining domains CDR1, CDR2, and CDR3. In some embodiments, the CDR1 comprises GSTFSDYG (SEQ ID NO: 100). In some embodiments, the CDR2 comprises an IDWNGEHT (SEQ ID NO: 101). In some embodiments, the CDR3 comprises AADALPYTVRKYNY (SEQ ID NO: 102). In some embodiments, the ISVD is humanized. In some embodiments, the ISVD comprises SEQ ID NO 77.

In some aspects, polypeptides are provided that comprise GSTFSDYG (SEQ ID NO: 100), IDWNGEHT (SEQ ID NO: 101), and AADALPYTVRKYNY (SEQ ID NO: 102). In another aspect, there is provided a polypeptide comprising an ISVD provided herein. In some embodiments, the polypeptide comprises a second binding moiety. In some embodiments, the second binding moiety binds to CD8 or another different target. In some embodiments, the second binding moiety is also ISVD. In some embodiments, the polypeptide comprises a detectable label. In some embodiments, the polypeptide comprises a therapeutic agent.

In some aspects, provided are compositions comprising an ISVD provided herein or a polypeptide provided herein.

In some aspects, provided are pharmaceutical compositions comprising an ISVD provided herein or a polypeptide provided herein and a pharmaceutically acceptable carrier.

In some aspects, methods of treating a CD 8-associated disease or disorder in a subject are provided. In some embodiments, the method comprises administering to the subject a pharmaceutical composition described herein. In some embodiments, the disease or disorder is cancer.

Various aspects and embodiments of the invention are described in further detail below.

Drawings

FIG. 1 depicts the NMR spectrum of lipid 1.

Fig. 2A and 2B depict LC-MS spectra of lipid 1.

FIG. 3 depicts the NMR spectrum of lipid 2.

Fig. 4A and 4B depict LC-MS spectra of lipid 2.

FIG. 5 depicts the NMR spectrum of lipid 3.

FIG. 6 depicts the NMR spectrum of lipid 4.

Fig. 7A and 7B depict LC-MS spectra of lipid 4.

Fig. 8A depicts lipid 2 and lipid 6LNP pKa (TNS). Fig. 8B depicts lipid 5 and lipid 7LNP pKa (TNS).

Fig. 9A depicts the hydrodynamic diameters of lipid 2 and lipid 6-derived LNPs. Fig. 9B depicts the polydispersity (dynamic light scattering) of lipid 2 and lipid 6-derived LNPs.

Figure 10A depicts the hydrodynamic diameters of lipid 5 and lipid 7-derived LNPs. Fig. 10B depicts the polydispersity (dynamic light scattering) of lipid 5 and lipid 7-derived LNPs.

FIGS. 11A-11D depict in vitro T cell transfection of GFP mRNA using lipid 2 and lipid 6-derived LNPs: % GFP+ cells (FIG. 11A), GFP Mean Fluorescence Intensity (MFI) (FIG. 11B),% Cy5-GFP+ cells (FIG. 11C), and Cy5-GFP MFI, E.T cell viability (FIG. 11D).

Figures 12A-12E depict in vitro T cell transfection of GFP mRNA using lipid 5 and lipid 7-derived LNP: % GFP+ cells (FIG. 12A), GFP Mean Fluorescence Intensity (MFI) (FIG. 12B),% Cy5-GFP+ cells (FIG. 12C), cy5-GFP MFI (FIG. 12D), T cell viability (FIG. 12E).

Figure 13A depicts%gfp+ (translated) human CD 8T cells 24h post transfection. Figure 13B depicts% Cy5+ (binding) human CD 8T cells 24h post transfection.

Figure 14A depicts% live human CD 8T cells 24h post transfection. Fig. 14B depicts human ifnγ measured from cell culture supernatant 24h after transfection.

FIG. 15A depicts% TTR-023+ (anti-CD 20 CAR) CD 8T cells 24h after transfection with mRNA LNP. FIG. 15B depicts% TTR-023+ (anti-CD 20 CAR) CD 4T cells after 24h transfection with mRNA LNP.

Figure 16A depicts% cd69+cd8 cells after 24h transfection with anti-CD 20 CAR mRNA LNP. Figure 16B depicts% cd69+cd4T cells after 24h transfection with anti-CD 20 CAR mRNA LNP.

Figure 17 depicts human ifnγ secretion from T cells 24h after LNP transfection with anti-CD 20 CAR mRNA.

FIG. 18A depicts% GFP+ of CD 8T cells after 24h transfection with 2.5ug/mL Cy5/GFP mRNA (transfection/translation). FIG. 18B depicts the% GFP+ (transfection/translation) average fluorescence intensity (MFI) of CD 8T cells after 24h transfection with 2.5ug/mL Cy5/GFP mRNA.

FIG. 19A depicts% Cy5+ (binding) of CD 8T cells after 24h transfection with 2.5ug/mL Cy5/GFP mRNA. FIG. 19B depicts the Cy5 (binding) Mean Fluorescence Intensity (MFI) of CD 8T cells after 24h transfection with 2.5ug/mL of Cy5/GFP mRNA.

FIG. 20A depicts% GFP+ (transfected/translated) CD8 cells of human CD3 cells transfected with 2.5ug/mL Cy5/GFP mRNA LNP for 24 h. FIG. 20B depicts% GFP+ (transfected/translated) CD4 cells of human CD3 cells transfected with 2.5ug/mL Cy5/GFP mRNA LNP for 24 h.

FIG. 21A depicts% Cy5+ (binding) CD8 cells of human CD3 cells transfected with 2.5ug/mL Cy5/GFP mRNA LNP for 24 h. FIG. 21B depicts% Cy5+ (binding) CD4 cells of human CD3 cells transfected with 2.5ug/mL Cy5/GFP mRNA LNP for 24 h.

FIG. 22A depicts% CD69+CD8 cells of human CD3 cells transfected with 2.5ug/mL Cy5/GFP mRNA LNP for 24 h. FIG. 22B depicts% CD69+CD4 cells of human CD3 cells transfected with 2.5ug/mL Cy5/GFP mRNA LNP for 24 h.

FIG. 23 depicts human IFNγ secretion from human CD3 cells transfected with 2.5 μg/mL Cy5/GFP mRNA LNP for 24 h.

FIG. 24A depicts%mCherry+CD8T cells transfected in whole blood at 2.5 μg/mL mCherry mRNA LNP for 24 h. FIG. 24B depicts%mCherry+CD4T cells transfected in whole blood at 2.5 μg/mL mCherry mRNA LNP for 24 h.

FIG. 25A depicts%mCherry+B cells transfected in whole blood at 2.5 μg/mL mCherry mRNA LNP for 24 h. FIG. 25B depicts%mCherry+NK cells transfected in whole blood at 2.5 μg/mL mCherry mRNA LNP for 24 h.

FIG. 26A depicts%mCherry+ granulocytes transfected in whole blood at 2.5 μg/mL mCherry mRNA LNP for 24 h. FIG. 26B depicts% CD69+CD8T cells transfected in whole blood at 2.5ug/mL mCherry mRNA LNP for 24 h. FIG. 26C depicts% CD69+CD4T cells transfected in whole blood at 2.5ug/mL mCherry mRNA LNP for 24 h.

FIGS. 27A and 27B depict the time course of in vivo reprogramming of CD8+ T cells and CD4+ T cells with CD 3-targeted mCherry LNP in blood, respectively. Each symbol represents one mouse. Open symbols represent buffer control treated mice and filled symbols represent mCherry LNP treated mice. Circles represent 24h, triangles represent 48h, and diamonds represent 96h. Fig. 27C and 27D depict the time course of in vivo reprogramming of cd8+ T cells and cd4+ T cells in the liver, respectively. Each symbol represents one mouse. Open symbols represent buffer control treated mice and filled symbols represent mCherry LNP treated mice. Circles represent 24h, triangles represent 48h, and diamonds represent 96h. FIGS. 27E and 27F depict the time course of in vivo reprogramming of CD8+ T cells and CD4+ T cells with CD 3-targeted mCherry LNP in the spleen, respectively. Each symbol represents one mouse. Open symbols represent buffer control treated mice and filled symbols represent mCherry LNP treated mice. Circles represent 24h, triangles represent 48h, and diamonds represent 96h.

FIG. 28 depicts minimum expression of mCherry in liver myeloid cells and Coulter cells after 24h treatment with CD 3-targeted mCherry LNP. Each symbol represents one mouse. Open symbols represent buffer control treated mice and filled symbols represent mCherry LNP treated mice.

Fig. 29A depicts in vivo reprogramming 24h after the first dose of LNP expressing mCherry in blood. Each symbol represents one mouse. Open circles are cd4+ T cells expressing mCherry, and open squares are cd8+ T cells expressing mCherry. Fig. 29B depicts in vivo reprogramming 24h after the first dose of TTR-023 expressing LNP in blood. Each symbol represents one mouse. Open circles are cd4+ T cells expressing anti-CD 20 CAR, and open squares are cd8+ T cells expressing anti-CD 20 CAR.

Fig. 30A-30E depict in vivo reprogramming in blood (fig. 30A), spleen (fig. 30B), liver (fig. 30C), bone marrow (fig. 30D), and thymus (fig. 30E) 40h after a second dose of LNP expressing TTR-023. Each symbol represents one mouse. Open circles are cd4+ T cells expressing anti-CD 20 CAR, and open squares are cd8+ T cells expressing anti-CD 20 CAR.

Fig. 31A-31E depict in vivo reprogramming in blood (fig. 31A), spleen (fig. 31B), liver (fig. 31C), bone marrow (fig. 31D), and thymus (fig. 31E) 40h after a second dose of LNP expressing mCherry. Each symbol represents one mouse. Open circles are cd4+ T cells expressing mCherry, and open squares are cd8+ T cells expressing mCherry.

Fig. 32 depicts the dosing and exsanguination regimen of the PK study.

FIG. 33 depicts mRNA concentrations calculated based on transformed DiI-C18 (3) -DS measurements from mouse serum samples.

FIG. 34A depicts% DiR+CD4T cells after incubation with 2.5 μg/mL mRNA LNP for 2 h. FIG. 34B depicts DiR Mean Fluorescence Intensity (MFI) CD 4T cells after incubation with 2.5 μg/mL mRNA LNP for 2 h.

FIG. 35A depicts% DiR+CD8T cells after incubation with 2.5 μg/mL mRNA LNP for 2 h. FIG. 35B depicts DiR Mean Fluorescence Intensity (MFI) CD 8T cells after incubation with 2.5. Mu.g/mL mRNA LNP for 2 h.

FIG. 36A depicts% mCherry+CD4T cells after incubation with 2.5 μg/mL mRNA LNP for 24 h. FIG. 36B depicts% mCherry+CD8T cells after incubation with 2.5 μg/mL mRNA LNP for 24 h.

FIG. 37A depicts the hydrodynamic diameters of lipid 5, lipid 8 and DLn-MC3-DMA derived LNPs. FIG. 37B depicts the polydispersity (dynamic light scattering) of lipid 5, lipid 8 and DLn-MC3-DMA derived LNPs.

FIGS. 38A-38E depict in vitro T cell transfection of GFP mRNA using lipid 5, lipid 8 and DLn-MC3-DMA derived LNP: % GFP+ cells (FIG. 38A), GFP Mean Fluorescence Intensity (MFI) (FIG. 38B),% Cy5-GFP+ cells (FIG. 38C), cy5-GFP MFI (FIG. 38D) and T cell viability (FIG. 38E).

FIG. 39 depicts the NMR spectrum of lipid 5.

Fig. 40A and 40B depict LC-MS spectra of lipid 5.

FIG. 41 depicts the NMR spectrum of lipid 6.

Fig. 42A and 42B depict LC-MS spectra of lipid 6.

FIG. 43 depicts the NMR spectrum of lipid 7.

Fig. 44A and 44B depict LC-MS spectra of lipid 7.

Figure 45A depicts the hydrodynamic diameters of lipid 8 and lipid 5-derived LNPs. Fig. 45B depicts the polydispersity (dynamic light scattering) of lipid 8 and lipid 5-derived LNPs.

FIGS. 46A-46E depict in vitro T cell transfection of GFP mRNA using lipid 8 and lipid 5 (O and N) -derived LNPs: % GFP+ cells (FIG. 46A), GFP Mean Fluorescence Intensity (MFI) (FIG. 46B),% Cy5-GFP+ cells (FIG. 46C), cy5-GFP MFI (FIG. 46D), T cell viability (FIG. 46E).

FIG. 47 depicts the structure of various Fab, VHH (Nb), scFv, fab-ScFv and Fab-VHH hybrids.

FIG. 48A depicts the NMR spectrum of lipid 9. Fig. 48B and 48C depict mass spectra and LC chromatograms of lipid 9.

Fig. 49A depicts NMR spectra of lipid 10. Fig. 49B and 49C depict mass spectra and LC chromatograms of lipid 10.

FIG. 50A depicts the NMR spectrum of lipid 11. Fig. 50B and 50C depict mass spectra and LC chromatograms of lipid 11.

FIG. 51A depicts the NMR spectrum of lipid 12. Fig. 51B and 51C depict mass spectra and LC chromatograms of lipid 12.

Fig. 52A depicts NMR spectra of lipid 13. Fig. 52B and 52C depict mass spectra and LC chromatograms of lipid 13.

Figure 53A depicts the hydrodynamic Diameters (DLS) of lipid 5 and lipid 8 before and after insertion of the antibody conjugate. Fig. 53B depicts polydispersity (DLS) before and after insertion of an antibody conjugate. Fig. 53C and 53D depict LNP surface charges (zeta potential, DLS) before and after insertion of antibody conjugates in pH 5.5MES and pH 7.4HEPES buffers.

Fig. 54A-54E depict in vitro T cell transfection of GFP mRNA using lipid 5 and lipid 8-derived LNPs: % gfp+ cells (fig. 54A), GFP Mean Fluorescence Intensity (MFI) (fig. 54B),% dii+ cells (fig. 54C), and DiI MFI (fig. 54D) and T cell viability (fig. 54E).

FIG. 55A depicts hydrodynamic Diameters (DLS) of lipid 5, lipid 8 and DLn-MC3-DMA before and after insertion of antibody conjugates. Fig. 55B depicts polydispersity (DLS) before and after insertion of an antibody conjugate. Fig. 55C depicts LNP surface charge (zeta potential, DLS) in pH 5.5MES and pH 7.4HEPES buffer prior to insertion of antibody conjugate. Fig. 55D depicts accessible RNA content and RNA encapsulation efficiency.

FIGS. 56A-56E depict in vitro T cell transfection of GFP mRNA using lipid 5, lipid 8 and DLn-MC3-DMA derived LNP: % gfp+ cells (fig. 56A), GFP Mean Fluorescence Intensity (MFI) (fig. 56B),% dii+ cells (fig. 56C), and DiI MFI (fig. 56D) and T cell viability (fig. 56E).

FIG. 57A depicts the hydrodynamic Diameter (DLS) of lipid 5 formulations stored at 4deg.C or after storage at-80deg.C; the formulations were frozen by placing in a-80 ℃ refrigerator or snap frozen in liquid nitrogen. Fig. 57B depicts formulation polydispersity (DLS) before and after frozen storage.

FIGS. 58A-58E depict in vitro T cell transfection and T cell viability of GFP mRNA produced from lipid 5LNP formulations stored at 4deg.C or after storage at-80deg.C; the formulations were frozen by placing in a-80 ℃ refrigerator or snap frozen in liquid nitrogen. % gfp+ cells (fig. 58A), GFP Mean Fluorescence Intensity (MFI) (fig. 58B),% dii+ cells (fig. 58C), and DiI MFI (fig. 58D) and T cell viability (fig. 58E).

FIGS. 59A-59T depict the results of in vivo reprogramming of immune cells with CD3 targeted DiI/GFP LNP at a dose of 0.3mg/kg after 2.5%24 or 48h with DMG, DPG or DSG-PEG or after 1.5% or 2.5%24h with DPPE or DSPE. Each symbol represents one mouse. Open circles are expressed cd4+ T cells and open squares are expressed cd8+ T cells; (fig. 59A) GFP in the blood, (fig. 59B) in the liver, (fig. 59C) in the lung, (fig. 59D) in the spleen, (fig. 59E) in the bone marrow; (fig. 59F) GFP MFI in blood, (fig. 59G) in liver, (fig. 59H) in lung, (fig. 59I) in spleen, (fig. 59J) in bone marrow; (fig. 59K)% DiI in blood, (fig. 59L) in liver, (fig. 59M) in lung, (fig. 59N) in spleen, (fig. 59O) in bone marrow; diI MFI (fig. 59P) in blood, (fig. 59Q) in liver, (fig. 59R) in lung, (fig. 59S) in spleen and (fig. 59T) in bone marrow.

Figures 60A-60T depict in vivo reprogramming results with 0.3mg/kg CD3, CD8 antibody/nanobody targeted DiI/GFP LNP with lipid 5 after 1.5% or 2.5%24h with DMG, DPG. Each symbol represents one mouse. Open circles are expressed cd4+ T cells and open squares are expressed cd8+ T cells; (60A) GFP in% in blood, (60B) in liver, (60C) in lung, (60D) in spleen, (60E) in bone marrow; (60F) GFP MFI in blood, (60G) in liver, (60H) in lung, (60I) in spleen, (60J) in bone marrow; (60K) % DiI in blood, (60L) in liver, (60M) in lung, (60N) in spleen, (60O) in bone marrow; (60P) DiI MFI in blood, (60Q) in liver, (60R) in lung, (60S) in spleen, (60T) in bone marrow.

Fig. 61A-61T depict in vivo reprogramming results with 0.3mg/kg CD8, CD11A, CD4 nanobody or CD4 antibody targeted DiI/GFP LNP with lipid 5 after 1.5%24h with DMG or DPG. Each symbol represents one mouse. Open circles are expressed cd4+ T cells and open squares are expressed cd8+ T cells; (61A) GFP in% in blood, (61B) in liver, (61C) in lung, (61D) in spleen, (61E) in bone marrow; (61F) GFP MFI in blood, (61G) in liver, (61H) in lung, (61I) in spleen, (61J) in bone marrow; (61K) % DiI in blood, (61L) in liver, (61M) in lung, (61N) in spleen, (61O) in bone marrow; (61P) DiI MFI in blood, (61Q) in liver, (61R) in lung, (61S) in spleen, (61T) in bone marrow.

FIGS. 62A-62S depict in vivo reprogramming comparing 0.1mg/kg of ionizable lipids (DLn-MC 3-DMA, lipid 5, and lipid 8) with CD3 (hsp 34) antibody targeted DiI/GFP LNP after 1.5%24h with DPG-PEG. Each symbol represents one mouse. Open circles are expressed cd4+ T cells and open squares are expressed cd8+ T cells; (62A) GFP in% in blood, (62B) in liver, (62C) in lung, (62D) in spleen, (62E) in bone marrow; (62F) GFP MFI in blood, (62G) in liver, (62H) in lung, (62I) in spleen, (62J) in bone marrow; (62K) % DiI in blood, (62L) in liver, (62M) in lung, (62N) in spleen, (62O) in bone marrow; (62P) DiI MFI in blood, (62Q) in liver, (62R) in lung, (62S) in spleen, (62T) in bone marrow.

Figures 63A to 63T depict in vivo reprogramming with 0.3mg/kg CD7 VHH/nanobody targeting DiI/GFP LNP of lipid 5 after 1.5% or 2.5%24h with DMG, DPG. Each symbol represents one mouse. Open circles are expressed cd4+ T cells and open squares are expressed cd8+ T cells; (63A) % GFP in blood, (63B) in liver, (63C) in lung, (63D) in spleen, (63E) in bone marrow; (63F) GFP MFI in blood, (63G) in liver, (63H) in lung, (63I) in spleen, (63J) in bone marrow; (63K) % DiI in blood, (63L) in liver, (63M) in lung, (63N) in spleen, (63O) in bone marrow; (63P) DiI MFI in blood, (63Q) in liver, (63R) in lung, (63S) in spleen, (63T) in bone marrow.

FIG. 64A depicts% GFP transfection of co-cultured T cells and NK cells after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for approximately 24 h. FIG. 64B depicts GFP expression levels in terms of Mean Fluorescence Intensity (MFI) for co-cultured T cells and NK cells after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for approximately 24 h. Fig. 64C depicts% DiI uptake by co-cultured T cells and NK cells after incubation with targeted LNP inserted Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 64D depicts% DiI uptake by Mean Fluorescence Intensity (MFI) of co-cultured T cells and NK cells after incubation with targeted LNP inserted Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

FIG. 65A depicts SDS-PAGE of SP34-hlam DS (containing WT interchain disulfide bonds) Fab conjugates produced by reduction at different TCEP concentrations prior to conjugation. FIG. 65B depicts SDS-PAGE of SP34-hlam NoDS (without interchain disulfide bonds, e.g., C-to-S mutations in HC and LC) Fab conjugates produced by reduction at different TCEP concentrations prior to conjugation. FIG. 65C depicts R8 RP-HPLC chromatograms of hSP34-hlam DS Fab and Fab conjugates produced with 0.025mM TCEP reduction conditions prior to conjugation. FIG. 65D depicts R8 RP-HPLC chromatograms of hSP34-hlam NoDS Fab and Fab conjugates produced with various TCEP reduction conditions prior to conjugation. FIG. 65E depicts% GFP transfection of T cells at 2.5ug/mL mRNA after incubation with targeted LNP inserted with Fab at various densities for approximately 24 h. FIG. 65F depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP, inserted with Fab at various densities, at 2.5ug/mL mRNA for approximately 24 h.

FIG. 66A depicts SDS-PAGE of TS2/18.1 and 9.6 (containing WT interchain disulfide bonds) Fab conjugates produced by reduction at different TCEP concentrations prior to conjugation. Left: TS2/18.1; right: 9.6. FIG. 66B depicts SDS-PAGE of TS2/18.1, 9.6 and TRX2 NoDS Fab as well as Fab conjugates produced by reduction at different TCEP concentrations prior to conjugation. FIG. 66C depicts R8 RP-HPLC chromatograms of TS2/18.1DS and NoDS Fab conjugates produced with various TCEP reduction conditions prior to conjugation. FIG. 66D depicts R8 RP-HPLC chromatograms of 9.6 and TRX2 NoDS Fab conjugates produced with various TCEP reduction conditions prior to conjugation.

FIG. 67A depicts% GFP transfection of T cells after incubation with targeted LNP at 2.5ug/mL mRNA for approximately 24h, with Fab inserted after the targeted LNP gives the density of the highest transfection level evaluated. Fig. 67B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP at density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 67C depicts ifnγ secretion from T cells in supernatant after incubation with targeted LNP inserted Fab at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

FIG. 68A depicts% GFP transfection of T cells after incubation with targeted LNP at 2.5ug/mL mRNA for approximately 24h, with Fab inserted after the targeted LNP gives the density of the highest transfection level evaluated. Fig. 68B depicts GFP expression levels of CD 8T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24h, with Fab inserted.

FIG. 69A depicts% GFP transfection of T cells after incubation with targeted LNP alone or together at the same density as single targeting conditions at 2.5ug/mL mRNA for about 24 h. FIG. 69B depicts GFP expression levels in terms of Mean Fluorescence Intensity (MFI) for T cells after incubation with targeted LNP alone or together with Fab inserted at the same density as single targeting conditions at 2.5ug/mL mRNA for about 24 h. Fig. 69C depicts ifnγ secretion from T cells in supernatant after incubation with targeted LNP alone or together with Fab inserted at the same density as single targeting conditions at 2.5ug/mL mRNA for about 24 h.

FIG. 70A depicts% GFP transfection of T cells after incubation with targeted LNP inserted with Fab or Fab-ScFv at a density giving the highest level of transfection evaluated at 2.5ug/mL mRNA for approximately 24 h. FIG. 70B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP inserted with Fab or Fab-ScFv at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 70C depicts ifnγ secretion from T cells in supernatant after incubation with targeted LNP inserted Fab or Fab-ScFv at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

Figure 71A depicts% GFP transfection of T cells after incubation with targeted LNP at 2.5ug/mL mRNA for approximately 24h, with Fab inserted after the targeted LNP was given the density of the highest transfection level evaluated. Fig. 71B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP at density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 71C depicts ifnγ secretion from T cells in supernatant after incubation with targeted LNP inserted Fab at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

Figure 72A depicts% GFP transfection of T cells after incubation with targeted LNP inserted with Fab and Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 72B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP, inserted Fab and Nb at a density giving the highest transfection level evaluated, for about 24h at 2.5ug/mL mRNA. Fig. 72C depicts ifnγ secretion from T cells in supernatant after incubation with targeted LNP inserted with Fab and Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

Fig. 73A depicts% GFP transfection of T cells after incubation with targeted LNP inserted with Fab and Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 73B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP inserted with Fab and Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 73C depicts ifnγ secretion from T cells in supernatant after incubation with targeted LNP inserted with Fab and Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

Figure 74A depicts% GFP transfection of T cells after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 74B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

Fig. 75A depicts% GFP transfection of CD 8T cells after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest transfection level assessed at 2.5ug/mL mRNA for about 24 h. Fig. 75B depicts% GFP transfection of CD 4T cells after incubation with targeted LNP at 2.5ug/mL mRNA for approximately 24 hours, with Fab or Nb inserted after targeting LNP at a density giving the highest transfection level assessed. Fig. 75C depicts GFP expression levels of CD 8T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP at 2.5ug/mL mRNA for approximately 24h, with Fab or Nb inserted after targeting LNP at a density giving the highest transfection level assessed. Fig. 75D depicts GFP expression levels of CD 4T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP at 2.5ug/mL mRNA for approximately 24h, with Fab or Nb inserted after targeting LNP at a density giving the highest transfection level assessed.

FIG. 76A depicts% GFP transfection of T cells after incubation with targeted LNP inserted with Fab and Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for approximately 24 h. Fig. 76B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP inserted with Fab and Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

FIG. 77A depicts% GFP transfection of T cells after incubation with targeted LNP inserted with Fab and Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for approximately 24 h. Fig. 77B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP inserted with Fab and Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

Figure 78A depicts% GFP transfection of T cells after incubation with targeted LNP inserted with Fab and Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 78B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP, inserted Fab and Nb at a density giving the highest transfection level evaluated, for about 24h at 2.5ug/mL mRNA.

FIG. 79A depicts% GFP transfection of T cells after incubation with targeted LNP at 2.5ug/mL mRNA for approximately 24h, with Fab inserted after the targeted LNP gives the density of the highest transfection level evaluated. FIG. 79B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP, inserted with Fab at a density giving the highest transfection level evaluated, for about 24h at 2.5ug/mL mRNA. Fig. 79C depicts ifnγ secretion from T cells in supernatant after incubation with targeted LNP inserted Fab at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

FIG. 80A depicts% GFP transfection of CD 8T cells after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for approximately 24 h. FIG. 80B depicts% GFP transfection of CD 4T cells after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for approximately 24 h. Fig. 80C depicts GFP expression levels of CD 8T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP at 2.5ug/mL mRNA for approximately 24h, with Fab or Nb inserted after targeting LNP at a density giving the highest transfection level assessed. FIG. 80D depicts GFP expression levels of CD 4T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP, inserted with Fab or Nb at a density giving the highest transfection level evaluated, for about 24h at 2.5ug/mL mRNA. Fig. 80E depicts ifnγ secretion from T cells in supernatant after incubation with targeted LNP inserted Fab at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

FIG. 81A depicts% GFP transfection of T cells after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest level of transfection evaluated at 2.5ug/mL mRNA for approximately 24 h. FIG. 81B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP, inserted with Fab or Nb at a density giving the highest transfection level evaluated, for about 24h at 2.5ug/mL mRNA. Fig. 81C depicts ifnγ secretion from T cells after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

Fig. 82A depicts% GFP transfection of T cells after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 82B depicts GFP expression levels of T cells according to Mean Fluorescence Intensity (MFI) after incubation with targeted LNP inserted with Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h. Fig. 82C depicts ifnγ secretion from T cells after incubation with targeted LNP inserted Fab or Nb at a density giving the highest transfection level evaluated at 2.5ug/mL mRNA for about 24 h.

Figure 83A depicts the hydrodynamic Diameters (DLS) of lipid 2, lipid 6, lipid 12 and lipid 13 before and after insertion of the antibody conjugate. Fig. 83B depicts polydispersity (DLS) before and after insertion of an antibody conjugate. Fig. 83C depicts LNP surface charge (zeta potential, DLS) in pH 5.5MES and pH 7.4HEPES buffer prior to insertion of antibody conjugate. FIG. 83D depicts the percentage of accessible RNA and total RNA content (ug/mL).

Figures 84A-84E depict in vitro T cell transfection of GFP mRNA using lipid 2, lipid 6, lipid 12 and lipid 13-derived LNPs: % gfp+ cells (fig. 84A), GFP Mean Fluorescence Intensity (MFI) (fig. 84B),% dii+ cells (fig. 84C), and DiI MFI (fig. 84D) and T cell viability (fig. 84E).

Fig. 85A-85E depict in vitro T cell transfection of GFP mRNA using lipid 2, lipid 6, lipid 12 and lipid 13-derived LNPs: % gfp+ cells (fig. 85A), GFP Mean Fluorescence Intensity (MFI) (fig. 85B),% dii+ cells (fig. 85C), and DiI MFI (fig. 85D) and T cell viability (fig. 85E).

Detailed Description

The practice of the present invention employs, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, cell biology and biochemistry. Such techniques are explained in the literature, e.g. "Comprehensive Organic Synthesis" (b.m. Trost and i.fleming editions, 1991-1992); "Current protocols in molecular biology" (F.M. Ausubel et al, editions, 1987, and periodic updates); and "Current protocols in immunology" (J.E. Coligan et al, editions, 1991), each of which is incorporated herein by reference in its entirety. Various aspects of the invention are set forth in the sections below; however, aspects of the invention described in one particular section are not limited to any particular section.

I. Definition of the definition

In order to facilitate an understanding of the present invention, a number of terms and phrases are defined below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations used herein have their conventional meaning in the chemical and biological arts. The chemical structures and formulas set forth herein should be construed in accordance with standard rules of valences known in the chemical arts. In addition, for example, when the chemical group is a diradical, it is understood that the chemical group may be bonded to its adjacent atoms in the remainder of the structure in one or both directions, e.g., -OC (O) -may be interchangeable with-C (O) O-, or-OC (S) -may be interchangeable with-C (S) O-.

The terms "a" and "an" as used herein mean "one or more" and include plural unless the context is not appropriate.

The term "alkyl" as used herein refers to saturated straight or branched chain hydrocarbons, such as straight or branched chain groups of 1 to 12, 1 to 10 or 1 to 6 carbon atoms, referred to herein as C1-C12 alkyl, C1-C10 alkyl and C1-C6 alkyl, respectively. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2-dimethyl-1-butyl, 3-dimethyl-1-butyl, 2-ethyl-1-butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, and the like.

The term "alkylene" refers to a diradical of an alkyl group. An exemplary alkylene group is-CH 2CH2-.

The term "haloalkyl" refers to an alkyl group substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like.

The term "oxo" is art-recognized and refers to a "=o" substituent. For example, cyclopentane substituted with oxo groups is cyclopentanone.

The term "morpholino" refers to a substituent having the structure:

the term "piperidinyl" refers to a substituent having the structure:

in general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are taken appropriatelySubstitution of the substituents. Unless otherwise indicated, an "optionally substituted" group may have suitable substituents at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents at each position may be the same or different. The combinations of substituents envisioned under the present invention are preferably combinations of substituents that result in the formation of stable or chemically feasible compounds. In some embodiments, the optional substituents may be selected from: c (C) _1-6 Alkyl, cyano, halogen, -O-C _1-6 Alkyl, C _1-6 Haloalkyl, C _3-7 Cycloalkyl, 3-7 membered heterocyclyl, 5-6 membered heteroaryl and phenyl, wherein R ^a Is hydrogen or C _1-6 An alkyl group. In some embodiments, the optional substituents may be selected from: c (C) _1-6 Alkyl, halogen, -O-C _1-6 Alkyl and-CH ₂ N(R _a ) ₂ Wherein R is ^a Is hydrogen or C _1-6 An alkyl group.

The term "haloalkyl" refers to an alkyl group substituted with at least one halogen. For example, -CH ₂ F、-CHF ₂ 、-CF ₃ 、-CH ₂ CF ₃ 、-CF ₂ CF ₃ Etc.

The term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic, bridged (e.g., adamantyl) or spirocyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons derived from a cycloalkane, referred to herein as, for example, "C _4-8 Cycloalkyl groups). Exemplary cycloalkyl groups include, but are not limited to, cyclohexane, cyclopentane, cyclobutane, and cyclopropane. Unless otherwise indicated, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonate, phosphinate, sulfate, sulfide, sulfonamide, sulfonyl, or thiocarbonyl groups. In certain embodiments, cycloalkyl groups are not takenInstead, i.e. it is unsubstituted.

The terms "heterocyclyl" and "heterocyclic group" are art-recognized and refer to a saturated, partially unsaturated or aromatic 3-to 10-membered ring structure, alternatively a 3-to 7-membered ring, which ring structure includes one to four heteroatoms, such as nitrogen, oxygen and sulfur. The number of ring atoms in the heterocyclic group may be C _x -C _x Nomenclature designations where x is an integer designating the number of ring atoms. For example, C ₃ -C ₇ Heterocyclyl refers to a saturated or partially unsaturated 3-to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen and sulfur. Name "C ₃ -C ₇ "indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, including any heteroatoms occupying the ring atom positions. C (C) ₃ An example of a heterocyclic group is aziridinyl. The heterocycle may be, for example, a monocyclic, bicyclic, or other polycyclic ring system (e.g., fused, spiro, bridged bicyclic). The heterocycle may be fused to one or more aryl, partially unsaturated or saturated rings. Heterocyclic groups include, for example, biotinyl, chromene, dihydrofuryl, indolinyl, dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl, imidazolidinyl, isoquinolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxathiolanyl, oxazolidinyl, phenoxanyl (phenoxanyl), piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazolinyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydroquinolinyl, thiazolidinyl, thiolanyl, thiomorpholinyl, thiopyranyl, xanthenyl, lactones, lactams (such as azetidinone and pyrrolidone), sultam, sultones, and the like. Unless otherwise indicated, the heterocycle is optionally substituted at one or more positions with a moiety such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, oxo, phosphate, phosphonate, phosphinate, sulfate, sulfide Substituted with substituents such as a compound, sulfonamide, sulfonyl, and thiocarbonyl groups. In certain embodiments, the heterocyclyl is unsubstituted, i.e., it is unsubstituted.

The term "aryl" is art recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. The term "aryl" includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjacent rings (which are "fused rings"), wherein at least one ring is aromatic, and for example, the other ring or rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, and/or aryl. Unless otherwise indicated, an aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido, carboxylic acid, -C (O) alkyl, CO ₂ Alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moiety, -CF ₃ -CN, etc. In certain embodiments, the aromatic ring is substituted with halogen, alkyl, hydroxy, or alkoxy at one or more ring positions. In certain other embodiments, the aromatic ring is unsubstituted, i.e., it is unsubstituted. In certain embodiments, aryl is a 6-10 membered ring structure.

The term "heteroaryl" is art-recognized and refers to an aromatic group that includes at least one ring heteroatom. In certain instances, heteroaryl groups contain 1, 2, 3, or 4 ring heteroatoms. Representative examples of heteroaryl groups include pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and the like. Unless otherwise indicated, heteroaryl rings may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido, carboxylic acid, C (O) alkyl, -CO ₂ Alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclic groupAryl or heteroaryl moiety, -CF ₃ -CN, etc. The term "heteroaryl" also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjacent rings (which are "fused rings"), wherein at least one ring is heteroaromatic, e.g., other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, and/or aryl. In certain embodiments, the heteroaryl ring is substituted with halogen, alkyl, hydroxy, or alkoxy at one or more ring positions. In certain other embodiments, the heteroaryl ring is unsubstituted, i.e., it is unsubstituted. In certain embodiments, heteroaryl groups are 5 to 10 membered ring structures, alternatively 5 to 6 membered ring structures, which ring structures include 1, 2, 3, or 4 heteroatoms, such as nitrogen, oxygen, and sulfur.

The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., those of the general formula-N (R ¹⁰ )(R ¹¹ ) A moiety of the representation, wherein R ¹⁰ And R is ¹¹ Each independently represents hydrogen, alkyl, cycloalkyl, heterocyclyl, alkenyl, aryl, aralkyl or (CH) ₂ ) _m -R ¹² The method comprises the steps of carrying out a first treatment on the surface of the Or R is ¹⁰ And R is ¹¹ Together with the N atom to which they are attached, form a heterocyclic ring having from 4 to 8 atoms in the ring structure; r is R ¹² Represents aryl, cycloalkyl, cycloalkenyl, heterocycle or polycyclic; and m is zero or an integer in the range of 1 to 8. In certain embodiments, R ¹⁰ And R is ¹¹ Each independently represents hydrogen, alkyl, alkenyl or- (CH) ₂ ) _m -R ¹² 。

The term "alkoxy" or "alkoxy" is art-recognized and refers to an alkyl group as defined above having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, t-butoxy, and the like. An "ether" is two hydrocarbons covalently linked by oxygen. Thus, the substituent of the alkyl group which renders the alkyl group an ether is or is analogous to an alkoxy group, e.g. can be made up of-O-alkyl, -O-alkenyl, O-alkynyl, -O- (CH) ₂ ) _m -R ¹² Wherein m and R are ¹² As described above. The term "haloalkoxy"Refers to an alkoxy group substituted with at least one halogen. For example, -O-CH ₂ F、-O-CHF ₂ 、-O-CF ₃ Etc. In certain embodiments, haloalkoxy is alkoxy substituted with at least one fluorine group. In certain embodiments, haloalkoxy is alkoxy substituted with from 1 to 6, 1 to 5, 1 to 4, 2 to 4, or 3 fluoro groups.

(symbol)Indicating the attachment point.

The compounds of the present disclosure may contain one or more chiral centers and/or double bonds and thus exist as stereoisomers (e.g., geometric isomers, enantiomers or diastereomers). As used herein, the term "stereoisomer" consists of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbol "R" or "S", depending on the configuration of substituents around the stereogenic carbon atom. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated by "(±)" in nomenclature, but the skilled artisan will recognize that the structure may implicitly represent a chiral center. It is to be understood that the graphic depiction of a chemical structure (e.g., a general chemical structure) encompasses all stereoisomeric forms of the specified compound unless otherwise indicated.

The individual stereoisomers of the compounds of the invention may be prepared synthetically from commercially available starting materials containing asymmetric or stereocenters or by preparing racemic mixtures followed by resolution procedures well known to those of ordinary skill in the art. These splitting methods are exemplified by the following: (1) Attaching the enantiomeric mixture to a chiral auxiliary, separating the resulting diastereomeric mixture by recrystallization or chromatography, and releasing the optically pure product from the auxiliary; (2) forming a salt using an optically active resolving agent; or (3) directly separating the mixture of optical enantiomers on a chiral chromatographic column. The stereoisomeric mixtures may also be resolved into their constituent stereoisomers by well known methods, such as chiral phase gas chromatography, chiral phase high performance liquid chromatography, crystallization of compounds as chiral salt complexes or crystallization of compounds in chiral solvents. Further, the enantiomers may be separated using Supercritical Fluid Chromatography (SFC) techniques described in the literature. Still further, stereoisomers may be obtained from stereoisomerically pure intermediates, reagents and catalysts by well known asymmetric synthetic methods.

Geometric isomers may also be present in the compounds of the present invention. Sign symbolRepresents a bond that may be a single bond, a double bond, or a triple bond as described herein. The present invention encompasses various geometric isomers and mixtures thereof arising from arrangements of substituents around carbon-carbon double bonds or arrangements of substituents around carbocycles. Substituents around a carbon-carbon double bond are designated as being in the "Z" or "E" configuration, wherein the terms "Z" and "E" are used according to IUPAC standards. Unless otherwise indicated, structures describing double bonds encompass both the "E" and "Z" isomers.

Substituents around a carbon-carbon double bond may alternatively be referred to as "cis" or "trans", where "cis" means that the substituents are on the same side of the double bond and "trans" means that the substituents are on opposite sides of the double bond. The arrangement of substituents around a carbocycle is designated "cis" or "trans". The term "cis" means that the substituents are on the same side of the ring plane, and the term "trans" means that the substituents are on opposite sides of the ring plane. Mixtures of compounds in which substituents are placed on the same side and opposite sides of the ring plane are designated "cis/trans".

The present invention also includes isotopically-labeled compounds of the present invention, which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as correspondingly ² H、 ³ H、 ¹³ C、 ¹⁴ C、 ¹⁵ N、 ¹⁸ O、 ¹⁷ O、 ³¹ P、 ³² P、 ³⁵ S、 ¹⁸ F and F ³⁶ Cl。

Certain isotopically-labeled disclosed compounds (e.g., compounds labeled with 3H and 14C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and may be preferred in some circumstances. Isotopically-labeled compounds of the present invention can generally be prepared by procedures analogous to those disclosed in the examples herein by substituting a non-isotopically-labeled reagent with an isotopically-labeled reagent.

As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods of the invention. Such organisms are preferably mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably are humans.

As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, such that the composition is particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

As used herein, the term "pharmaceutically acceptable excipient" refers to any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions (such as, for example, oil/water or water/oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Remington's The Science and Practice of Pharmacy, 21 st edition, a.r. gennaro; lippincott, williams & Wilkins, baltimore, MD,2006.

As known to those skilled in the art, "salts" of the compounds of the present invention may be derived from inorganic or organic acids and inorganic or organic bases. Examples of acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid and the like. Other acids, such as oxalic acid, while not pharmaceutically acceptable per se, may be used to prepare salts which may be used as intermediates in obtaining the compounds of the invention and pharmaceutically acceptable acid addition salts thereof.

Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and formula NW ₄ ⁺ Compounds of (wherein W is C) _1-4 Alkyl), and the like.

Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethane sulfonate, fumarate, fluoroheptanoate (flufluoroheptanoate), glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmitate, pectate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include salts with suitable cations such as Na ⁺ 、NH ₄ ⁺ And NW ₄ ⁺ (wherein W is C _1-4 Alkyl), and the like.

Abbreviations as used herein include Diisopropylethylamine (DIPEA); 4-Dimethylaminopyridine (DMAP); tetrabutylammonium iodide (TBAI); 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC); benzotriazol-1-yl-oxy-tripyrrolidinylphosphonium hexafluorophosphate (PyBOP); 9-fluorenylmethoxycarbonyl (Fmoc); tetrabutyldimethylsilyl chloride (TBDMSCl); hydrogen Fluoride (HF); phenyl (Ph); bis (trimethylsilyl) amine (HMDS); dimethylformamide (DMF); dichloromethane (DCM); tetrahydrofuran (THF); high Performance Liquid Chromatography (HPLC); mass Spectrometry (MS); evaporative Light Scattering Detector (ELSD); electrospray (ES); nuclear magnetic resonance spectroscopy (NMR).

As used herein, the term "effective amount" refers to an amount of a compound (e.g., a nucleic acid, such as mRNA) sufficient to achieve a beneficial or desired result. An effective amount may be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration. The term effective amount may be considered to include a therapeutically and/or prophylactically effective amount of the compound.

The phrase "therapeutically effective amount" as used herein means an amount of a compound (e.g., a nucleic acid, such as an mRNA), material, or composition comprising a compound (e.g., a nucleic acid, such as an mRNA) that is effective to produce some desired therapeutic effect in at least one cell subset in a mammal (e.g., a human) or subject (e.g., a human subject) at a reasonable benefit/risk ratio applicable to any medical treatment.

The phrase "prophylactically effective amount" as used herein means an amount of a compound (e.g., a nucleic acid, such as an mRNA), material, or composition comprising a compound (e.g., a nucleic acid, such as an mRNA) that is effective to produce some desired prophylactic effect in at least one cell subset in a mammal (e.g., a human) or subject (e.g., a human subject) by reducing, minimizing, or eliminating the risk of developing a disorder, or reducing or minimizing the severity of a disorder at a reasonable benefit/risk ratio applicable to any medical treatment.

As used herein, the terms "treatment", "treatment" and "treatment" include any effect that results in the improvement of a condition, disease, disorder, etc., or the amelioration of a symptom thereof, such as reduction, alleviation, modulation, amelioration, or elimination.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

In the present application, where an element or component is said to be included in and/or selected from a list of enumerated elements or components, it is understood that the element or component may be any one of the enumerated elements or components, or the element or component may be selected from two or more of the enumerated elements or components.

Further, it is to be understood that elements and/or features of the compositions or methods described herein may be combined in various ways, whether explicit or implicit herein, without departing from the spirit and scope of the present application. For example, unless otherwise understood from the context, where a particular compound is mentioned, the compound may be used in various embodiments of the compositions of the application and/or in the methods of the application. In other words, within the present application, embodiments have been described and depicted in a manner that enables a clear and concise application to be written and drawn, but it is intended and understood that embodiments may be combined or separated in various ways without departing from the teachings of the application and one or more applications. For example, it is to be understood that all features described and depicted herein may be applicable to all aspects of one or more of the applications described and depicted herein.

It should be understood that unless otherwise understood from the context and use, the expression "at least one of … …" includes each/every single one of the objects listed after the expression and each combination of two or more of the listed objects. Unless otherwise understood from the context, the expression "and/or" in combination with three or more/three or more of the listed objects should be understood to have the same meaning.

Unless otherwise explicitly stated or otherwise understood from the context, the use of the terms "include," "comprising," "including," "having," "with," "containing," or "containing" (including grammatical equivalents thereof) shall generally be construed as open-ended and non-limiting, e.g., not excluding additional unrecited elements or steps.

The invention also includes the specific values themselves, where the term "about" is used prior to a value unless explicitly stated otherwise. As used herein, unless otherwise indicated or inferred, the term "about" refers to a variation of ±10% from the nominal value.

As used herein, unless otherwise indicated, the term "antibody" means any antigen binding molecule or molecular complex that comprises at least one Complementarity Determining Region (CDR) that specifically binds or interacts with a particular antigen. It is understood that the term encompasses whole antibodies (e.g., whole monoclonal antibodies) or fragments thereof, such as Fc fragments of antibodies (e.g., fc fragments of monoclonal antibodies) or antigen-binding fragments of antibodies (e.g., antigen-binding fragments of monoclonal antibodies), including whole antibodies, antigen-binding fragments, or Fc fragments that have been modified or engineered. Examples of antigen binding fragments include Fab, fab ', (Fab') ₂ Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). The term also encompasses immunoglobulin single variable domains, such as nanobodies (e.g., V _HH )。

As used herein, an "antibody that binds to X" (i.e., X is a particular antigen) or an "anti-X antibody" is an antibody that specifically recognizes antigen X.

As used herein, "entrapped interchain disulfide bonds" or "interchain entrapped disulfide bonds" refer to disulfide bonds on a polypeptide that are not readily accessible to, or effectively "entrapped" in, the hydrophobic region of the polypeptide by a water-soluble reducing agent, such that it is neither available for use as a reducing agent nor for conjugation with other hydrophilic PEG. The embedded interchain disulfide bonds are further described in WO 2017096361A1, which is incorporated by reference in its entirety.

As used herein, the specificity of targeted delivery of LNP is defined by the ratio between the% of the desired immune cell type that receives the nucleic acid being delivered (e.g., mid-target delivery) and the% of the unwanted immune cell type that is not intended to be the target of the delivery but that receives the nucleic acid being delivered (e.g., off-target delivery). For example, specificity is higher when more desired immune cells receive the delivered nucleic acid while fewer unwanted immune cells receive the delivered nucleic acid. The specificity of targeted delivery of LNP can also be defined as the ratio of the amount of nucleic acid delivered to a desired immune cell (e.g., mid-target delivery) to the amount of nucleic acid delivered to an undesired immune cell (e.g., off-target delivery). The specificity of delivery may be determined using any suitable method. As a non-limiting example, the expression level of a nucleic acid in a desired immune cell type may be measured and compared to the expression level of a nucleic acid in a different immune cell type not intended to be the target of the delivery.

As used herein, in some embodiments, the reference LNP is an LNP that does not have an immune cell targeting group but is otherwise identical to the LNP tested. In some other embodiments, the reference LNP is an LNP having a different ionizable cationic lipid but otherwise identical to the LNP tested. In some embodiments, the reference LNP comprises D-Lin-MC3-DMA as an ionizable cationic lipid (which is different from the ionizable cationic lipid in the tested LNP), but otherwise identical to the tested LNP.

As used herein, a humanized antibody is an antibody that is of wholly or partially non-human origin and whose protein sequence has been modified to replace certain amino acids, for example amino acids that occur at one or more corresponding positions in the framework regions of VH and VL domains in the sequence of an antibody from a human, to increase its similarity to an antibody naturally produced in the human, in order to avoid or minimize an immune response in the human. For example, the variable domain of a non-human antibody of interest can be combined with the constant domain of a human antibody using genetic engineering techniques. The constant domains of humanized antibodies are, in many cases, human CH and CL domains.

As used herein, the term "structural lipid" refers to sterols, and also refers to lipids containing a sterol moiety.

It should be understood that the order of steps or the order in which certain actions are performed is immaterial so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

Substituents are disclosed in groups or ranges throughout the specification. In particular, it is intended that the description include each individual sub-combination of the members of such groups and ranges. For example, the term "C _1-6 Alkyl "is specifically intended to disclose C alone ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ 、C ₆ 、C ₁ -C ₆ 、C ₁ -C ₅ 、C ₁ -C ₄ 、C ₁ -C ₃ 、C ₁ -C ₂ 、C ₂ -C ₆ 、C ₂ -C ₅ 、C ₂ -C ₄ 、C ₂ -C ₃ 、C ₃ -C ₆ 、C ₃ -C ₅ 、C ₃ -C ₄ 、C ₄ -C ₆ 、C ₄ -C ₅ And C ₅ -C ₆ An alkyl group. By way of further example, integers in the range of 0 to 40 are specifically intended to disclose 0, 1, 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 and 40 individually, and integers in the range of 1 to 20 are specifically intended to disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 individually.

The use of any and all examples, or exemplary language (e.g., "such as" or "comprising") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Throughout the specification, where compositions and kits are described as having, comprising or including specific components, or where processes and methods are described as having, comprising or including specific steps, it is contemplated that there are additionally compositions and kits of the invention consisting essentially of or consisting of the recited components, as well as processes and methods according to the invention consisting essentially of or consisting of the recited processing steps.

Typically, unless otherwise indicated, the indicated percentages of the composition are by weight. Further, if a variable does not have an accompanying definition, then the previous definition of the variable is followed.

Immunoglobulin single variable domains

In some embodiments, the immune cell targeting group of an LNP as described herein comprises an immunoglobulin single variable domain, such as a nanobody.

The term "immunoglobulin single variable domain" (ISV), which is used interchangeably with "single variable domain", defines an immunoglobulin molecule in which an antigen binding site is present on and formed from a single immunoglobulin domain. This allows the immunoglobulin single variable domain to be compared to a "conventional" immunoglobulin (e.g., monoclonal antibody) or fragment thereof (e.g., fab ', F (ab') ₂ scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V _H ) And a light chain variable domain (V _L ) Interact to form an antigen binding site. In this case V _H And V _L The Complementarity Determining Regions (CDRs) of both will contribute to the antigen binding site, i.e., a total of 6 CDRs will be involved in the formation of the antigen binding site. In view of the above definition, conventional 4-chain antibodies (e.g., igG, igM, igA, igD or IgE molecules; known in the art) or Fab, F (ab') ₂ Antigen binding domains of fragments, fv fragments (such as disulfide-linked Fv or scFv fragments), or diabodies (all known in the art) will generally not be considered immunoglobulin single variable domains, as in these casesIn the case of an epitope corresponding to an antigen, the binding to the corresponding epitope of the antigen usually does not take place via a (single) immunoglobulin domain, but via a pair of (associated) immunoglobulin domains (e.g. light and heavy chain variable domains) which are jointly bound to the epitope of the corresponding antigen, i.e. via the V of the immunoglobulin domains _H -V _L The pairing occurs.

In contrast, an immunoglobulin single variable domain is capable of specifically binding to an epitope of an antigen without pairing with another immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain consists of a single V _H Single V _HH Or a single V _L Domain formation. Thus, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

Thus, the single variable domain can be a light chain variable domain sequence (e.g., V _L Sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., V _H Sequence or V _HH Sequence) or a suitable fragment thereof; so long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit consisting essentially of a single variable domain such that a single antigen binding domain need not interact with another variable domain to form a functional antigen binding unit).

The immunoglobulin single variable domain may be, for example, a heavy chain ISV, such as V _H 、V _HH Comprising camelized V _H Or humanized V _HH . In one embodiment, it is V _HH Comprising camelized V _H Or humanized V _HH . The heavy chain ISV may be derived from conventional four-chain antibodies or heavy chain antibodies.

For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence suitable for use as a single domain antibody), "dAb" or dAb (or an amino acid sequence suitable for use as a dAb) orISV (as defined herein, and including but not limited to V _HH ) The method comprises the steps of carrying out a first treatment on the surface of the Other singly variable structuresA domain, or any suitable fragment thereof.

In particular, the immunoglobulin single variable domain may beISV (e.g. V) _HH Including humanized V _HH Or camelized V _H ) Or a suitable fragment thereof. Note that: />Is a registered trademark of Ablynx N.V]。

“V _HH Domain "(also known as V) _HH 、V _HH Antibody fragments and V _HH Antibodies) have been described initially as "heavy chain antibodies" (i.e., "antibodies without light chains"; hamers-Casterman et al 1993Nature 363:446-448) antigen binding immunoglobulin variable domains. The term "V" has been selected _HH Domains "so as to associate these variable domains with the heavy chain variable domains present in conventional 4-chain antibodies (which are referred to herein as" V " _H Domain ") and a light chain variable domain (which is referred to herein as" V ") found in conventional 4-chain antibodies _L Domains ") are distinguished. With respect to V _HH For further description, refer to the review article of Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302).

For the terms "dAb" and "domain antibody", reference is made, for example, to Ward et al 1989 (Nature 341:544), holt et al 2003 (Trends Biotechnol.21:484); and other published patent applications such as WO 2004/068820, WO 2006/030220, WO 2006/003388 and Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention, because they are not of mammalian origin, the single variable domains may be derived from certain shark species (e.g. so-called "IgNAR domains", see e.g. WO 2005/18629).

Typically, the production of immunoglobulins involves immunization of experimental animals, fusion of immunoglobulin-producing cells to produce hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins may be generated by screening a naive, immune or synthetic library, for example by phage display.

The production of immunoglobulin sequences such as VHH has been widely described in various publications, including WO 1994/04678, hamers-Casterman et al 1993 (Nature 363:446-448) and Muyledermans et al 2001 (Reviews in Molecular Biotechnology 74:277-302,2001). In these methods, a camelid is immunized with a target antigen in order to induce an immune response against the target antigen. The VHH library obtained from the immunization is further screened for VHH binding to target antigen.

In these cases, the production of antibodies requires purified antigen for immunization and/or screening. The antigen may be purified from natural sources or during recombinant production. Immunization and/or screening of immunoglobulin sequences may be performed using peptide fragments of such antigens.

Immunoglobulin sequences of different origins may be used herein, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. Furthermore, fully human, humanized or chimeric sequences may be used in the methods described herein. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, may be used herein, e.g., as described by Ward et al 1989 (Nature 341:544), WO 1994/04678, and Davis and Riechmann (1994, febs Lett.,339:285-290; and 1996, prot. Eng., 9:531-537). In addition, ISVs are fused to form multivalent and/or multispecific constructs (with respect to containing one or more V' s _HH Multivalent and multispecific polypeptides of domains and their preparation are also referred to in Conrath et al 2001 (J.biol. Chem., vol.276, 10.7346-7350), e.g., WO 1996/34103 and WO 1999/23221).

"humanized V _HH "comprising a polypeptide corresponding to naturally occurring V _HH The amino acid sequence of the domain has been "humanized" (i.e., by use in a conventional 4-chain antibody from a human (e.g., as indicated above)) V _H Substitution of one or more amino acid residues occurring at one or more corresponding positions in the domainChanging the naturally occurring V _HH Amino acid sequence of one or more amino acid residues in the amino acid sequence of the sequence, in particular in the framework sequence. This can be done in a manner known per se, which should be clear to the skilled person, for example based on prior art (e.g. WO 2008/020079). Also, it should be noted that such humanized V _HH Can be obtained in any suitable manner known per se and is therefore not strictly limited to polypeptides which have been obtained using polypeptides comprising naturally occurring VHH domains as starting materials.

"camel derived V _H "comprising a polypeptide corresponding to naturally occurring V _H The amino acid sequence of the domain has been "camelized" (i.e., by use in the V of (camelid) heavy chain antibodies _HH Substitution of one or more amino acid residues occurring at one or more corresponding positions in the domain for naturally occurring V from conventional 4-chain antibodies _H One or more amino acid residues in the amino acid sequence of the domain). This can be done in a manner known per se, which should be clear to the skilled person, for example based on the description of the prior art (e.g. Davies and Riechman 1994,FEBS 339:285;1995,Biotechnol.13:475;1996,Prot.Eng.9:531; and Riechman 1999,J.Immunol.Methods 231:25). Such "camelized" substitution inserts are formed and/or present in V, as defined herein _H -V _L The amino acid position of the interface and/or at a so-called camelid tag residue (see e.g.WO 1994/04678, davies and Riechmann (1994 and 1996, supra)). In one embodiment, as a production or design of camelized V _H V of the starting material or origin of (C) _H The sequence is V from a mammal _H Sequences, e.g. human V _H Sequences, e.g. V _H 3 sequence. However, it should be noted that such camelized V _H Can be obtained in any suitable manner known per se and is therefore not strictly limited to the use of a composition comprising naturally occurring V _H A polypeptide obtained from a polypeptide having a domain as a starting material.

The structure of an immunoglobulin single variable domain sequence may be considered to be composed of four framework regions ("FR"), which are referred to in the art and herein as "framework region 1" ("FR 1"), respectively; "frame region 2" ("FR 2"); "frame region 3" ("FR 3"); and "frame region 4" ("FR 4"); the framework region is interrupted by three complementarity determining regions ("CDRs") referred to in the art and herein, respectively, as "complementarity determining region 1" ("CDR 1"); "complementarity determining region 2" ("CDR 2"); and "complementarity determining region 3" ("CDR 3").

In such immunoglobulin sequences, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences should be apparent to the skilled artisan, e.g., based on standard handbooks and further disclosures and the prior art mentioned herein.

The framework sequences are immunoglobulin framework sequences or framework sequences that have been derived (e.g., by humanization or camelization) from immunoglobulin framework sequences (suitable combinations). For example, the framework sequence may be derived from a light chain variable domain (e.g., V _L Sequence) and/or heavy chain variable domains (e.g., V _H Sequence or V _HH Sequence) of the sequence. In a particular aspect, the framework sequence is that which has been derived from V _HH The framework sequences of the sequences (where the framework sequences may optionally have been partially or fully humanised) or are conventional V which have been camelised _H Sequences (as defined herein).

In particular, the framework sequences present in the ISV sequences described herein may contain one or more tag residues (as defined herein) such that the ISV sequence isISV, e.g. like V _HH Including humanized V _HH Or camelized V _H . Non-limiting examples of (suitable combinations of) such framework sequences will become apparent from the further disclosure herein.

V _H Domain and V _HH The total number of amino acid residues in the domain will typically range from 110 to 120, typically between 112 and 115. However, should beIt is noted that smaller and longer sequences may also be suitable for the purposes described herein.

It should be noted, however, that ISVs described herein are not limited with respect to the origin of the ISV sequence (or the nucleotide sequence used to express it), and with respect to the manner in which the ISV sequence or nucleotide sequence is generated or obtained (or has been generated or obtained). Thus, an ISV sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In a specific but non-limiting aspect, an ISV sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semisynthetic sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequences, particularly partially or fully humanized V _HH Sequence), "camelized" (as defined herein) immunoglobulin sequence (particularly camelized V) _H Sequence), and ISVs that have been obtained by techniques such as: affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences that are well known to the skilled artisan; or any suitable combination of any of the foregoing.

Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semisynthetic sequence, and may be a sequence isolated from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence that has been isolated from a library (particularly an expression library), a nucleotide sequence that has been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers, or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se, for example.

In general, the number of the devices used in the system,ISV (especially V) _HH Sequences, including (partially) humanized V _HH Sequence and camelization V _H Sequences) may be characterized by the presence of one or more "tag residues" (as described herein) in one or more framework sequences (also as described further herein). Thus, in general, ->ISV can be defined as an immunoglobulin sequence having the following (general) structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein one or more tag residues are as further defined herein.

In particular the number of the elements to be processed,ISVs may be immunoglobulin sequences having the following (general) structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein the framework sequences are as further defined herein.

More particularly, it is possible to provide,ISVs may be immunoglobulin sequences having the following (general) structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein: one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are selected from the marker residues mentioned in table 2A below.

Table 2A:markers in ISVResidues->

/>

In one embodiment, the immunoglobulin single variable domain has certain amino acid substitutions in the framework regions that are effective to prevent or reduce binding of a so-called "pre-existing antibody" to the polypeptide. Such ISVs have been described in WO2015/173325, wherein (i) the amino acid residue at position 112 is one of K or Q; and/or (ii) the amino acid residue at position 89 is T; and/or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V.

Polypeptides

The immunoglobulin single variable domain may form part of a protein or polypeptide, which may comprise or consist essentially of one or more (at least one) immunoglobulin single variable domains, and may optionally further comprise one or more other amino acid sequences (all optionally linked via one or more suitable linkers). The term "immunoglobulin single variable domain" may also encompass such polypeptides. The one or more immunoglobulin single variable domains may be used as binding units in such proteins or polypeptides, which may optionally contain one or more other amino acids that may be used as binding units, in order to provide monovalent, multivalent or multispecific polypeptides of the invention, respectively (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, see also concoth et al 2001 (j. Biol. Chem. 276:7346) and e.g. WO 1996/34103, WO 1999/23221 and WO 2010/115998).

The polypeptide may comprise or consist essentially of an immunoglobulin single variable domain, as outlined above. Such polypeptides are also referred to herein as monovalent polypeptides.

The term "multivalent" indicates the presence of multiple ISVs in a polypeptide. In one embodiment, the polypeptide is "bivalent", i.e. comprises or consists of two ISVs. In one embodiment, the polypeptide is "trivalent", i.e. comprises or consists of three ISVs. In another embodiment, the polypeptide is "tetravalent", i.e., comprises or consists of four ISVD. Thus, the polypeptide may be "bivalent", "trivalent", "tetravalent", "pentavalent", "hexavalent", "heptavalent", "octavalent", "nonavalent" or the like, i.e. the polypeptide comprises or consists of two, three, four, five, six, seven, eight, nine etc. ISVs, respectively. In one embodiment, the multivalent ISV polypeptide is trivalent. In another embodiment, the multivalent ISV polypeptide is tetravalent. In yet another embodiment, the multivalent ISV polypeptide is pentavalent.

In one embodiment, the multivalent ISV polypeptide may also be multispecific. The term "multispecific" refers to binding to a plurality of different target molecules (also referred to as antigens). Thus, the multivalent ISV polypeptide may be "bispecific", "trispecific", "tetraspecific", etc., i.e. may bind to two, three, four, etc., different target molecules, respectively.

For example, the polypeptide may be bispecific-trivalent, such as a polypeptide comprising or consisting of three ISVs, wherein two ISVs bind to a first target and one ISV binds to a second target different from the first target. In another example, the polypeptide may be trispecific-tetravalent, such as a polypeptide comprising or consisting of four ISVs, one of which binds to a first target, two of which bind to a second target different from the first target, and one of which bind to a third target different from the first and second targets. In yet another example, the polypeptide may be trispecific-pentavalent, such as a polypeptide comprising or consisting of five ISVs, two of which bind to a first target, two of which bind to a second target different from the first target, and one of which bind to a third target different from the first and second targets.

In one embodiment, the multivalent ISV polypeptide may also be multi-paratope. The term "multiple paratope" refers to binding to a plurality of different epitopes on the same target molecule (also referred to as antigen). Thus, the multivalent ISV polypeptide may be "biparatopic", "tricaratopic" or the like, i.e. capable of binding to two, three or the like different epitopes on the same target molecule, respectively.

In another aspect, a polypeptide of the invention comprising or consisting essentially of one or more immunoglobulin single variable domains (or suitable fragments thereof) may further comprise one or more other groups, residues, moieties or binding units. Such other groups, residues, portions, binding units, or amino acid sequences may or may not provide other functions to the immunoglobulin single variable domain (and/or polypeptide in which it is present), and may or may not alter the properties of the immunoglobulin single variable domain.

For example, such other groups, residues, moieties or binding units may be one or more additional amino acids, such that the compound, construct or polypeptide is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, the one or more other groups, residues, moieties or binding units are immunoglobulins. Even more preferably, the one or more additional groups, residues, moieties or binding units are selected from the group consisting of domain antibodies, amino acids suitable for use as domain antibodies, single domain antibodies, amino acids suitable for use as single domain antibodies, "dabs", amino acids suitable for use as dabs or nanobodies.

Alternatively, such groups, residues, moieties or binding units may be, for example, chemical groups, residues, moieties, which may or may not themselves have biological and/or pharmacological activity. For example, but not limited to, such groups may be linked to the one or more immunoglobulin single variable domains so as to provide a "derivative" of the immunoglobulin single variable domain.

In another embodiment, the additional residues may be effective to prevent or reduce binding of a so-called "pre-existing antibody" to the polypeptide. For this purpose, the polypeptides and constructs may contain a C-terminal extension (X) n (SEQ ID NO: 150) (wherein n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (preferably 1 or 2, such as 1), and each X is independently selected, preferably independently selected, from the (preferably naturally occurring) amino acid residues of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I), for which reference is made to WO 2012/175741). Thus, the polypeptide may further comprise a C-terminal extension (X) n (SEQ ID NO: 151), wherein n is 1 to 5, such as 1, 2, 3, 4 or 5, and wherein X is a naturally occurring amino acid, preferably not cysteine.

In the polypeptides described above, the one or more immunoglobulin single variable domains and the one or more groups, residues, moieties or binding units may be directly linked to each other and/or via one or more suitable linkers or spacers. For example, where the one or more groups, residues, moieties or binding units are amino acids, the linker may also be an amino acid, such that the resulting polypeptide is a fusion protein or fusion polypeptide.

As used herein, the term "linker" refers to a peptide that fuses two or more ISVs together to form a single molecule. The use of linkers to join two or more (poly) peptides is well known in the art. Other exemplary peptide linkers are shown in table 2B. One type of commonly used peptide linker is known as a "Gly-Ser" or "GS" linker. These are linkers consisting essentially of glycine (G) and serine (S) residues, and typically comprise one or more repeats of a peptide motif, such as a GGGGS (SEQ ID NO: 154) motif (e.g., having the formula (Gly-Gly-Gly-Gly-Ser) n (SEQ ID NO: 152), where n may be 1, 2, 3, 4, 5, 6, 7 or greater. Some common examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 157), the 15GS linker (n=3), and the 35GS linker (n=7). See, for example, chen et al 2013 (adv. Drug Deliv. Rev.65 (10): 1357-1369) and Klein et al 2014 (Protein Eng. Des. Sel.27 (10): 325-330).

Table 2B: the linker sequence ("ID" refers to SEQ ID NO as used herein)

In one aspect, the present disclosure also relates to such amino acid sequences and/or nanobodies that can bind to and/or be directed against CD8 and comprise CDR sequences as generally further defined herein; suitable fragments thereof; and polypeptides comprising or consisting essentially of one or more such nanobodies and/or suitable fragments. In some aspects, the disclosure relates to nanobodies having SEQ ID NO. 77. In particular, in some particular aspects, the present disclosure provides:

i) An amino acid sequence that is directed against CD8 and has at least 80%, preferably at least 85% (e.g. 90% or 95% or more) sequence identity with at least one amino acid sequence of SEQ ID No. 77;

II) cross-block the binding of the amino acid sequence of SEQ ID NO:77 to CD8 and/or at least compete with the amino acid sequence of SEQ ID NO:77 for binding to CD 8;

such amino acid sequences may be as further described herein (and may be, for example, nanobodies); as well as polypeptides of the present disclosure comprising one or more such amino acid sequences (which may be as further described herein), in particular bispecific (or multispecific) polypeptides as described herein, and nucleic acid sequences encoding such amino acid sequences and polypeptides. Such amino acid sequences and polypeptides do not include any naturally occurring ligands.

In some embodiments, CD8 is derived from a mammal, such as a human. In one particular but non-limiting aspect, the present disclosure relates to an amino acid sequence for CD8 comprising:

a) The amino acid sequence of SEQ ID NO. 77;

b) An amino acid sequence having at least 80% amino acid identity to at least one amino acid sequence of SEQ ID NO. 77, or

c) An amino acid sequence having a 3, 2 or 1 amino acid difference from at least one amino acid sequence of SEQ ID NO 77;

or any suitable combination thereof.

In some embodiments, nanobodies against CD8 are disclosed that consist of 4 framework regions (FR 1 to FR4, respectively) and 3 complementarity determining regions (CDR 1 to CDR3, respectively). In some embodiments, in such nanobodies:

(I) CDR1 comprises or consists essentially of the amino acid sequence: the amino acid sequence of GSTFSDYG (SEQ ID NO: 100),

or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity to GSTFSDYG (SEQ ID NO: 100), wherein (1) any amino acid substitution is a conservative amino acid substitution; and/or (2) the amino acid sequence contains only amino acid substitutions and NO amino acid deletions or insertions compared to GSTFSDYG (SEQ ID NO: 100);

And/or from an amino acid sequence having a difference of 2 or only 1 amino acid from GSTFSDYG (SEQ ID NO: 100), wherein

Any amino acid substitution is a conservative amino acid substitution; and/or

In contrast to GSTFSDYG (SEQ ID NO: 100), the amino acid sequence contains only amino acid substitutions and NO amino acid deletions or insertions.

(II) CDR2 comprises or consists essentially of the amino acid sequence: the amino acid sequence of IDWNGEHT (SEQ ID NO: 101),

or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity to an idwneht (SEQ ID NO: 101), wherein (1) any amino acid substitution is a conservative amino acid substitution; and/or (2) the amino acid sequence contains only amino acid substitutions and NO amino acid deletions or insertions compared to the idwneht (SEQ ID NO: 101);

and/or from an amino acid sequence having a difference of 2 or only 1 amino acid from IDWNGEHT (SEQ ID NO: 101), wherein

Any amino acid substitution is a conservative amino acid substitution; and/or

In contrast to IDWNGEHT (SEQ ID NO: 101), the amino acid sequence contains only amino acid substitutions and NO amino acid deletions or insertions.

(III) CDR3 comprises or consists essentially of the amino acid sequence: AADALPYTVRKYNY (SEQ ID NO: 102),

Or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity to AADALPYTVRKYNY (SEQ ID NO: 102), wherein (1) any amino acid substitution is a conservative amino acid substitution; and/or (2) the amino acid sequence contains only amino acid substitutions and NO amino acid deletions or insertions compared to AADALPYTVRKYNY (SEQ ID NO: 102);

and/or from an amino acid sequence differing from AADALPYTVRKYNY (SEQ ID NO: 102) by 2 or only 1 amino acid, wherein

Any amino acid substitution is a conservative amino acid substitution; and/or

In contrast to AADALPYTVRKYNY (SEQ ID NO: 102), the amino acid sequence contains only amino acid substitutions and NO amino acid deletions or insertions.

CD8 nanobodies as disclosed herein may comprise one, two, or all three of the CDRs explicitly listed above. In some embodiments, the CD8 nanobody comprises:

CDR1: GSTFSDYG (SEQ ID NO: 100), based on IMGT name;

CDR2: IDWNGEHT (SEQ ID NO: 101), based on the IMGT name; and

CDR3: AADALPYTVRKYNY (SEQ ID NO: 102), based on the IMGT name.

In nanobodies of the disclosure comprising a combination of the above mentioned CDRs, each CDR may be replaced by a CDR selected from amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the mentioned CDR; wherein the method comprises the steps of

(1) Any amino acid substitution is preferably a conservative amino acid substitution; and/or

(2) The amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions compared to one or more of the above amino acid sequences;

and/or an amino acid sequence selected from the group consisting of having 3, 2 or only 1 (indicated in the previous paragraph) "amino acid differences" with one of the mentioned one or more CDR-above amino acid sequences, wherein:

(2) The amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions compared to one or more of the above amino acid sequences.

In one embodiment, the CD8 nanobody is BDSn:

anti-CD 8 BDSn Nb sequences (CDR 1, CDR2, CDR3 underlined, based on IMGT name):

EVQLVESGGGLVQAGGSLRLSCAASGSTFSDYGVGWFRQAPGKGREFVAD IDWNGEHTSYADSVKGRFATSRDNAKNTAYLQMNSLKPEDTAVYYCAADALPYTVR KYNYWGQGTQVTVSSGGCGGHHHHHH(SEQ ID NO:77)

in some embodiments, the CD8 nanobody of the disclosure is at 10 ^-5 To 10 ^-12 Molar (M) or less, preferably 10 ^-7 To 10 ^-12 Molar (M) or less, more preferably 10 ^-8 To 10 ^-12 Dissociation constant (KD) of mole/liter (M), and/or at least 10 ⁷ M ^-1 Preferably at least 10 ⁸ M ^-1 More preferably at least 10 ⁹ M ^-1 (e.g. at least 10 ¹² M ^-1 ) In particular with CD8 with a KD of less than 500nM, preferably less than 200nM, more preferably less than 10nM, such as less than 500. Mu.M. The KD and KA values for vWF of nanobodies of the disclosure can be determined in a manner known per se, for example using the assays described herein. More generally, the nanobodies described herein preferably have a dissociation constant for vWF as described in this paragraph.

In general, it should be noted that the term nanobody as used herein in its broadest sense is not limited to a particular biological source or a particular method of preparation. For example, as will be discussed in more detail below, nanobodies may be obtained by: (1) By isolating the VHH domain of a naturally occurring heavy chain antibody; (2) By expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) By "humanizing" (as described below) naturally occurring VHH domains or by expressing nucleic acids encoding such humanized VHH domains; (4) Naturally occurring VH domains from any animal species, particularly mammalian species (e.g. from a human), by "camelization" (as described below), or by expression of nucleic acids encoding such camelized VH domains; (5) By "camelization" of "domain antibodies" or "dabs" as described by Ward et al (supra), or by expression of nucleic acids encoding such camelized VH domains; (6) Synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences are used; (7) Preparing nucleic acid encoding nanobody by using nucleic acid synthesis technique, and then expressing the nucleic acid thus obtained; and/or (8) by any combination of the foregoing. Suitable methods and techniques for performing the foregoing will be apparent to those skilled in the art based on the disclosure herein, and include, for example, the methods and techniques described in more detail below.

In some embodiments, the CD8 nanobodies of the disclosure do not have an amino acid sequence that is identical (i.e., has a degree of 100% sequence identity) to the amino acid sequence of a naturally occurring VH domain (e.g., an amino acid sequence of a naturally occurring VH domain from a mammal, particularly from a human).

One class of CD8 nanobodies of the disclosure comprises nanobodies having an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VHH domain but which has been "humanized" (i.e. by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence with one or more amino acid residues occurring at one or more corresponding positions in the VH domain of a conventional 4-chain antibody from a human (e.g. indicated above). It should be noted that such humanized CD8 nanobodies of the present disclosure may be obtained in any suitable manner known per se (i.e. as indicated at points (1) - (8) above), and are therefore not strictly limited to polypeptides that have been obtained using polypeptides comprising naturally occurring VHH domains as starting materials.

Another class of CD8 nanobodies of the disclosure comprises nanobodies having an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VH domain that has been "camelized" (i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody with one or more amino acid residues that occur at one or more corresponding positions in the VHH domain of a heavy chain antibody). This can be done in a manner known per se, which should be clear to the skilled person, for example based on the further description below. Reference is also made to WO 94/04678. Such camelisation may take place preferentially at amino acid positions present at the VH-VL interface and at so-called camelid tag residues (see also e.g. WO 94/04678), as also mentioned below. In some embodiments, the VH domain or sequence used as a starting material or origin for the production or design of the camelized nanobody is a VH sequence from a mammal, such as a human VH sequence. It should be noted that such camelized nanobodies of the present disclosure may be obtained in any suitable manner known per se and are therefore not strictly limited to polypeptides that have been obtained using polypeptides comprising naturally occurring VH domains as starting materials.

For example, both "humanization" and "camelization" may be performed by: the nucleotide sequences encoding such naturally occurring VHH domains or VH domains, respectively, are provided and then one or more codons in the nucleotide sequences are altered in a manner known per se such that the new nucleotide sequences encode the humanized or camelized nanobody of the disclosure, respectively, and the nucleotide sequences thus obtained are then expressed in a manner known per se in order to provide the desired nanobody. Alternatively, the amino acid sequences of the desired humanized or camelized nanobodies of the present disclosure may be designed based on the amino acid sequences of naturally occurring VHH domains or VH domains, respectively, and then synthesized de novo using per se known peptide synthesis techniques. Furthermore, based on the amino acid sequence or nucleotide sequence of the naturally occurring VHH domain or VH domain, respectively, the nucleotide sequence encoding the desired humanized or camelized nanobody can be designed and then synthesized de novo using nucleic acid synthesis techniques known per se, after which the nucleotide sequence thus obtained can be expressed in a manner known per se in order to provide the desired nanobody.

Other suitable means and techniques for obtaining nanobodies and/or nucleotide sequences and/or nucleic acids encoding the same (starting from (amino acid sequences of) naturally occurring VH domains or preferably VHH domains) and/or from nucleotide sequences and/or nucleic acid sequences encoding the same) should be apparent to the skilled person and may for example comprise combining one or more amino acid sequences and/or nucleotide sequences from naturally occurring VH domains (such as one or more FR and/or CDR) with one or more amino acid sequences and/or nucleotide sequences from naturally occurring VHH domains (such as one or more FR or CDR) in a suitable manner in order to provide nanobodies (nucleotide sequences or nucleic acids encoding the same). Also provided are compounds and constructs, particularly proteins and polypeptides, comprising or consisting essentially of at least one such amino acid sequence and/or nanobody (or suitable fragment thereof) of the disclosure, and optionally further comprising one or more other groups, residues, moieties or binding units. In some embodiments, such other groups, residues, portions, binding units, or amino acid sequences may or may not provide other functions to the amino acid sequence and/or nanobody (and/or compounds or constructs in which it is present), and may or may not alter the properties of the amino acid sequence and/or nanobody.

The present disclosure also encompasses any polypeptide of the present disclosure that has been glycosylated at one or more amino acid positions, generally depending on the host used to express the polypeptide. The polypeptide may comprise an amino acid sequence of a CD8 nanobody of the disclosure fused to at least one other amino acid sequence at its amino terminus, at its carboxy terminus, or at both its amino terminus and at its carboxy terminus. Such other amino acid sequences may comprise at least one other nanobody, so as to provide a polypeptide comprising at least two (e.g., three, four, or five) nanobodies, wherein the nanobodies may optionally be linked via one or more linker sequences (as defined herein). The polypeptide comprising a CD8 nanobody and one or more additional nanobodies of the disclosure is a multivalent polypeptide. In multivalent polypeptides, the two or more nanobodies may be the same or different. For example, the two or more nanobodies in a multivalent polypeptide:

the antigen may be directed against the same part or epitope of the antigen or against two or more different parts or epitopes of the antigen; and/or

May be directed against different antigens;

or a combination thereof.

Thus, bivalent polypeptides, for example:

may comprise two identical nanobodies;

a first nanobody that may comprise a first part or epitope against an antigen and a second nanobody that is against the same part or epitope against the antigen or against another part or epitope of the antigen;

or may comprise a first nanobody directed against a first antigen and a second nanobody directed against a second antigen different from the first antigen;

whereas trivalent polypeptides of the invention are for example:

three identical or different nanobodies that may comprise identical or different portions or epitopes to the same antigen;

two identical or different nanobodies directed against identical or different portions or epitopes on a first antigen and a third nanobody directed against a second antigen different from said first antigen; or alternatively

A first nanobody against a first antigen, a second nanobody against a second antigen different from the first antigen, and a third nanobody against a third antigen different from the first and second antigens may be included.

CD8 nanobodies and polypeptides as disclosed herein may also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, e.g., for prophylactic and/or therapeutic purposes (e.g., as gene therapy). For this purpose, the nucleotide sequences encoding the CD8 nanobodies or polypeptides as disclosed herein may be introduced into the cells or tissues in any suitable manner, e.g. as such (e.g. using liposomes) or after they have been inserted into a suitable gene therapy vector (e.g. derived from a retrovirus such as an adenovirus or a parvovirus such as an adeno-associated virus). As will also be clear to the skilled artisan, such gene therapy may be performed in vivo and/or in situ in the patient's body by administering a nucleic acid of the invention or a suitable gene therapy vector encoding the nucleic acid to the patient or to a specific cell or specific tissue or organ of the patient; alternatively, suitable cells (typically taken from the body of the patient to be treated, such as transplanted lymphocytes, bone marrow aspirates or tissue biopsies) may be treated with the nucleotide sequences of the invention in vitro and then (re) introduced into the body of the patient as appropriate. All of these can be performed using Gene Therapy vectors, techniques and delivery systems well known to the skilled artisan, culver, k.w. "Gene Therapy",1994,p.xii,Mary Ann Liebert,Inc, publishers, new York, n.y.; giordano, nature F Medicine 2 (1996), 534-539; schaper, circ. Res.79 (1996), 911-919; anderson, science 256 (1992), 808-813; verma, nature 389 (1994), 239; isner, lancet 348 (1996), 370-374; muhlhauser, circ. Res.77 (1995), 1077-1086; onodera, blood 91; (1998), 30-36; verma, gene Ther.5 (1998), 692-699; nabel, ann.N.Y. Acad.Sci.811 (1997), 289-292; verzeletti, hum. Gene Ther.9 (1998), 2243-51; wang, nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; U.S. patent No. 5,580,8591; U.S. patent No. 5,589,5466; or Schaper, current Opinion in Biotechnology 7 (1996), 635-640. For example, in situ expression of ScFv fragments (Afanasieva et al, gene Ther.,10,1850-1859 (2003)) and diabodies (Blanco et al, J.Immunol,171,1070-1077 (2003)) has been described in the art.

Thus, also provided are nucleic acid sequences encoding CD8 nanobodies as described herein, and expression constructs and host cells comprising the nucleic acid sequences.

Methods of using the CD8 nanobodies and polypeptides of the disclosure are also disclosed.

In some embodiments, polypeptides comprising CD8 nanobodies may be used in lipid nanoparticles of the present disclosure to deliver nucleic acids to immune cells, as described herein. In some embodiments, the CD8 nanobodies and polypeptides of the disclosure can be used to treat a disorder or disease in a subject in need thereof. In some embodiments, such conditions or diseases include, but are not limited to, cancer, infection, immune disorders, autoimmune diseases.

In some embodiments, polypeptides comprising CD8 nanobodies may be used in imaging agents. In some embodiments, the imaging agent allows detection of human CD8, which is a specific biomarker found on the surface of a subset of T cells for diagnostic imaging of the immune system. Imaging of CD8 allows detection of T cell localization in vivo. The change in T cell localization may reflect the progression of the immune response and may occur over time due to various therapeutic treatments or even disease states. In some embodiments, it is used to image T cell localization for immunotherapy.

In addition, CD8 plays a role in activating downstream signaling pathways important for activating cytolytic T cells that function to clear viral pathogens and provide immunity against tumors. CD8 positive T cells recognize short peptides presented within the mhc i protein of antigen presenting cells. In some embodiments, polypeptides comprising CD8 nanobodies can enhance signaling through T cell receptors and enhance a subject's ability to clear viral pathogens and respond to tumor antigens. Thus, in some embodiments, the antigen binding constructs provided herein can be agonists and can activate CD8 targets.

Ionizable cationic lipids

Provided herein are ionizableCationic lipids, which can be used to produce lipid nanoparticle compositions to facilitate delivery of payloads (e.g., nucleic acids, such as DNA or RNA, such as mRNA) placed therein to cells, e.g., mammalian cells, e.g., immune cells. The ionizable cationic lipids have been designed to be capable of delivering nucleic acids (e.g., mRNA) intracellularly to the cytoplasmic compartments of the target cell type and rapidly degrading into non-toxic components. The complex function of the ionizable cationic lipid is facilitated by interactions between the ionizable lipid head group, the hydrophobic "acyl tail" group, and the chemistry and geometry of the linker connecting the head group and the acyl tail group. Typically, the ionizable amine head group has a pK of _a Designed to be in the range of 6-8, such as between 6.2-7.4 or between 6.5-7.1, so that it remains strongly cationic under acidic formulation conditions (e.g., pH 4-pH 5.5), neutral at physiological pH (7.4), and cationic in early and late endosomal compartments (e.g., pH 5.5-pH 7). The acyl tail groups play a key role in the fusion of the lipid nanoparticle with endosomal membranes and membrane destabilization by structural perturbation. The three-dimensional structure of the acyl tail (determined by its length, unsaturation, and site), and the relative sizes of the head and tail groups, are believed to play a role in promoting membrane fusion, and thus in lipid nanoparticle endosomal escape (a critical requirement for cytosolic delivery of nucleic acid payloads). The linker linking the head and acyl tail groups is designed to be degraded by a physiologically ubiquitous enzyme (e.g., esterase or protease) or by acid-catalyzed hydrolysis.

In one aspect, the present invention provides compounds represented by formula I:

or a salt thereof, wherein:

R ¹ and R is ² Independently C _1-3 Alkyl, or R ¹ And R is ² Forms together with the nitrogen atom an optionally substituted piperidinyl or morpholinyl group;

Y is selected from the group consisting of-O-, -OC (O) -, -OC (S) -and-CH ₂ -；

X ¹ 、X ² 、X ³ And X ⁴ Is hydrogen or X ¹ And X ² Or X ³ And X ⁴ Independently together form oxo;

n is 0 or 3;

o and p are independently integers selected from 2-6;

wherein the compound is not a compound selected from the group consisting of:

or a salt thereof. />

In certain embodiments, o and p may be 2. In certain embodiments, o and p may be 3. In other embodiments, o and p may be 4. In some embodiments, o and p may be 5. In other embodiments, o and p may be 6.

In certain embodiments, X ¹ And X ² Can together form oxo, and X ³ And X ⁴ Together forming oxo. In other embodiments, X ¹ 、X ² 、X ³ And X ⁴ May be hydrogen.

In some embodiments of the present invention, in some embodiments, Y may be selected from the group consisting of-O-, -OC (O) -, OC (S) -and-CH ₂ -. For example, in certain embodiments, Y may be-O-. In certain embodiments, Y may be-OC (O) -. In certain embodiments, Y may be-CH ₂ -. In certain embodiments, Y may be-OC (S) -.

In certain embodiments, R ¹ And R is ² Can be independently C _1-3 An alkyl group. In other embodiments, R ¹ And R is ² Can be-CH ₃ . In certain embodiments, R ¹ And R is ² is-CH ₂ CH ₃ . In certain embodiments, R ¹ And R is ² Is C ₃ An alkyl group.

In certain embodiments, n may be 0. In other embodiments, n may be 3.

Also provided herein, in part, are compounds represented by formula II:

or a salt thereof, wherein:

R ¹ and R is ² Independently C _1-3 Alkyl, or R ¹ And R is ² Forms together with the nitrogen atom an optionally substituted piperidinyl or morpholinyl group; y is selected from the group consisting of-O-, -OC (O) -, -OC (S) -and-CH ₂ -；

X ¹ 、X ² 、X ³ And X ⁴ Is hydrogen or X ¹ And X ² Or X ³ And X ⁴ Together forming oxo;

n is 0 to 4;

o is 1 and r is an integer selected from 3-8, or o is 2 and r is an integer selected from 1-8,

p is 1 and s is an integer selected from 3-8, or p is 2 and s is an integer selected from 1-8,

wherein,

when both o and p are 1, r and s are independently 4, 5, 7 or 8, and

when both o and p are 2, r and s are independently 1, 2, 4 or 5.

In certain embodiments, X ¹ And X ² Can together form oxo, and X ³ And X ⁴ Oxo may be formed together. In other embodiments, X ¹ 、X ² 、X ³ And X ⁴ May be hydrogen.

In certain embodiments, Y may be selected from the group consisting of-O-, -OC (O) -and-CH ₂ -. For example, in certain embodiments, Y may be-O-. In certain embodiments, Y may be-OC (O) -. In certain embodiments, Y may be-CH ₂ -. In certain embodiments, Y may be-OC (S) -.

In certain embodiments, R ¹ And R is ² Can be independently C _1-3 An alkyl group. In other embodiments, R ¹ And R is ² Can be-CH ₃ . In certain embodiments, R ¹ And R is ² Can be-CH ₂ CH ₃ . In some embodiments, R ¹ And R is ² May be C ₃ An alkyl group. In certain embodiments, R ¹ And R is ² Together with the nitrogen atom, form an optionally substituted piperidinyl group.

In certain embodiments, n may be 0. In other embodiments, n may be 3.

Provided herein, in part, are compounds selected from the group consisting of:

or a salt thereof.

Provided herein, in part, are compounds of the formula:

or a salt thereof.

Provided herein, in part, are compounds of the formula:

or a salt thereof.

Provided herein, in part, are compounds of the formula:

or a salt thereof.

Provided herein, in part, are compounds of the formula:

or a salt thereof.

Provided herein, in part, are compounds of the formula:

or a salt thereof.

Provided herein, in part, are compounds of the formula:

or a salt thereof.

Provided herein, in part, are compounds of the formula:

or a salt thereof.

In certain embodiments, the compound is a compound of formula III:

or a salt thereof, wherein:

X ¹ 、X ² 、X ³ And X ⁴ Is hydrogen or X ¹ And X ² Or X ³ And X ⁴ Together forming oxo; and is combined withAnd is also provided with

n is an integer selected from 0-4.

In certain embodiments, n may be 0. In other embodiments, n may be 3.

Also provided herein are compounds of the formula:

Or a salt thereof.

Scheme 1-synthetic scheme for preparing lipids of formula (I)

/>

Compounds of formula I can be prepared, for example, according to scheme 1. The hydroxy-functional protected propylene glycol is converted to the corresponding dimethylamino-functional ether (y=oxo) or ester (y=o-C (O)). The ether linkage is formed by the reaction of an alkyl halide with an alcohol in the presence of t-butyl ammonium iodide/NaOH in THF at 80 ℃. Ester bond formation utilizes treatment of acid functional dimethylamine with alcohol under carbodiimide activation (DCM, EDC, DIEPA, DMAP). Diol deprotection yields the vicinal diol intermediate which is subsequently converted to the corresponding ether-linked or ester-linked diacyl lipid by treatment with TBAI/NaOH and bromoacyl, or by carbodiimide-mediated activation of the carboxylic acid to form an ester linkage, respectively.

Scheme 2-synthetic scheme for preparing lipid compositions of formula (II)

Compounds of formula II can be prepared, for example, according to scheme 2. The synthetic procedure is outlined above for scheme 1; however, in scheme 2, di-or mono-unsaturated acyl groups may be used to obtain lipids of formula II.

In some embodiments, the ionizable cationic lipid used in the LNP of the present disclosure is selected from the lipids in table 1 or a combination thereof. In some embodiments, the ionizable cationic lipid is:

In some embodiments, the ionizable cationic lipid is not Dlin-MC3-DMA.

Lipid-immunocyte targeting group conjugates

As discussed herein, the LNP can be targeted to a particular cell type, such as an immune cell, e.g., a T cell, B cell, or Natural Killer (NK) cell. This may be achieved by using one or more lipids described herein. In addition, targeting may be enhanced by including targeting groups on the solvent accessible surface of the LNP particles. For example, a targeting group can include a member of a specific binding pair (e.g., an antibody-antigen pair, ligand-receptor pair, etc.). In certain embodiments, the targeting group is an antibody. Targeting can be performed, for example, by using the lipid-immune cell targeting group conjugates described herein.

Optionally, the targeting moiety is an antibody fragment without an Fc component. Previous attempts to target circulating immune cells with LNP have employed whole antibodies (WO 2016/189532 Al). Due to Fc conjugation, whole antibody-conjugated liposomes or lipid-based particles clear faster from circulation, reducing their likelihood of reaching target cells of interest (Harding et al (1997) Biochim Biophys. Acta 1327,181-192; sapra et al (2004) Clin Cancer Res 10,1100-1111; argnol et al, (1986) Proc Natl Acad Sci USA 83, 2699-2703). Liposomes targeted with antibody fragments retain their long circulating properties, such as liposomes targeted to EGFR (Mamot et al, (2005) Cancer Res 65, 11631-11638), erbB2 (Park et al (2002) Clin Cancer Res 8, 1172-1181), or EphA2 (Kamoun et al, 2019Nat.Biomed.Eng 3,264-280). In addition, lipid-based carriers can be prepared using micelle insertion methods that allow for the almost quantitative incorporation of antibody conjugates after their individual manufacture (Nellis et al (2005) Biotechnol Prog 21, 221-232) compared to the inefficiency of insertion when conjugating whole IgG (Ishida et al (1999) FEBS Lett.460, 129-133) or the need to complete conjugation directly on intact LNP (WO 2016/189532 Al). The scFv, fab or VHH fragment may also be conjugated directly to an activated PEG-lipid to form an insertable conjugate.

In certain embodiments, the targeting group may be a surface-binding antibody or surface-binding antigen-binding fragment thereof, which may allow for modulation of cell targeting specificity. This is particularly useful because highly specific antibodies can be raised against the epitope of interest at the desired targeting site. In one embodiment, a plurality of different antibodies can be incorporated and presented on the surface of the LNP, wherein each antibody binds to a different epitope on the same antigen or a different epitope on a different antigen. Such methods can increase the avidity and specificity of targeted interactions with specific target cells.

The targeting group or combination of targeting groups can be selected based on the desired localization, function or structural characteristics of a given target cell. For example, to target a T cell, population of T cells, or subpopulation of T cells, one or more antibodies or antigen-binding fragments or antigen-binding derivatives thereof that target T cells (e.g., via a T cell surface antigen) may be selected. Exemplary T cell surface antigens include, but are not limited to, for example, CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD39, CD69, CD103, CD137, CD45, T Cell Receptor (TCR) β, TCR- α/β, TCR- γ/δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, GL7, TLR2, TLR4, TLR5, and IL-15 receptor. For targeting NK cells or NK cell populations, one or more antibodies, antigen-binding fragments or antigen-binding derivatives thereof that target NK cells (e.g., via NK cell surface antigens) may be selected. Exemplary NK cell surface antigens include, but are not limited to, CD48, CD56, CD85a, CD85c, CD85d, CD85e, CD85f, CD85i, CD85j, CD158b2, CD161, CD244, CD16a, CD16b, IL-2 receptor, CD27, CD28, CD48, CD69, CD70, CD86, CD112, CD122, CD155, CD161, CD244, CD266, CD314/NKG2D, CD/NKP 44, CD337/NKP30. To target a B cell or population of B cells, one or more antibodies, antigen-binding fragments or antigen-binding derivatives thereof that target B cells (e.g., via a B cell antigen) may be selected. Exemplary B cell antigens include, but are not limited to, CD19 (for all B cells except plasma cells), CD19, CD25 and CD30 (for activated B cells), CD27, CD38, CD78, CD138 and CD319 (for plasma cells), CD20, CD27, CD40, CD80 and PDL-2 (for memory cells), notch2, CD1, CD21 and CD27 (for border region B cells), CD21, CD22 and CD23 (for follicular B cells), and CD1, CD5, CD21, CD24 and TLR4 (for regulatory B cells).

In certain embodiments, targeting can be performed, for example, by using the lipid-immune cell targeting group conjugates described herein. Exemplary lipid-immune cell targeting group conjugates can include compounds of formula IV,

[ lipid ] - [ optional linker ] - [ immune cell targeting group, e.g., T cell targeting molecule, e.g.,

anti-CD 2 antibody, anti-CD 3 antibody, anti-CD 7 antibody or anti-CD 8 antibody ]

(formula IV).

In some embodiments, the immune cell targeting group is a polypeptide and the lipid is conjugated to any position of the N-terminal, C-terminal, or middle portion of the polypeptide.

In certain embodiments, the targeting group or targeting molecule is a T cell targeting agent (e.g., an antibody) that binds to a T cell antigen selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T Cell Receptor (TCR) β, TCR- α/β, TCR- γ/δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor or IL-15 receptor. In certain embodiments, the T cell antigen may be CD2 and the targeting group may be, for example, an anti-CD 2 antibody. In certain embodiments, the T cell antigen may be CD3 and the targeting group may be, for example, an anti-CD 3 antibody. In certain embodiments, the T cell antigen may be CD4 and the targeting group may be, for example, an anti-CD 4 antibody. In certain embodiments, the T cell antigen may be CD5 and the targeting group may be, for example, an anti-CD 5 antibody. In certain embodiments, the T cell antigen may be CD7 and the targeting group may be, for example, an anti-CD 7 antibody. In certain embodiments, the T cell antigen may be CD8 and the targeting group may be, for example, an anti-CD 8 antibody. In certain embodiments, the T cell antigen may be tcrp and the targeting group may be, for example, an anti-tcrp antibody. In some embodiments, the antibody is a human or humanized antibody.

Exemplary CD2 binding agents may be antibodies selected from the group consisting of: 9.6 (https:// academic. Com/intim m/arc/10/12/1863/744536), 9-1 (https:// academic. Ou. Com/intim/arc/10/12/1863/744536), TS2/18.1.1 (ATCC HB-195), lo-CD2B (ATCC PTA-802), lo-CD2a/BTI-322 (U.S. Pat. No. 6849258B 1), sipilzumab (Sipilzumab)/MEDI-507 (U.S. Pat. No. 6849258B 1/en), 35.1 (ATCC HB-222), OKT11 (ATCC CRL-8027), RPA-2.1 (PCT publication WO 2020023559A 1), AF1856 (R)&D Systems)、MAB18562(R&D Systems)、MAB18561(R&D Systems)、MAB1856(R&D Systems), PAB30359 (Abnova Corporation), 10299-1 (Abnova Corporation) and antigen binding fragments thereof. In some embodimentsIn this case, the binding agent comprises a heavy chain variable domain (V _H ) And a light chain variable domain (V _L )：AF1856(R&D Sy stems)、MAB18562(R&D Systems)、MAB18561(R&D Systems)、MAB1856(R&D Systems), PAB30359 (Abnova Corporation) and 10299-1 (Abnova Corporation). In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H And V _L Heavy chain CDRs of a sequence ₁ 、CDR ₂ And CDR ₃ Light chain CDRs ₁ 、CDR ₂ And CDR ₃ It is determined according to Kabat (see, kabat et al, (1991) Sequen ces of Proteins of Immunological Interest, NIH Publication No.91-3242, bethesda), chothia (see, e.g., chothia C and Lesk A M, (1987), J.MOL.BIOL.196:901-917), macCalum (see, macCalum R M et al, (1996) J.MOL.BIOL.262:732-745), or any other CDR determining method known in the art: AF1856 (R) &D Systems)、MAB18562(R&D Systems)、MAB18561(R&D Systems)、MAB1856(R&D Systems), PAB30359 (Abnova Corporation) and 10299-1 (Abnova Corporation).

Exemplary CD2 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following clones: 9.6, 9-1, TS2/18.1.1, lo-CD2b, lo-CD2a, BTI-322, siprazumab, 35.1, OKT11, RPA-2.1, SQB-3.21, LT2, TS1/8, UT329, 4F22, OX-34, UQ2/42, MU3, U7.4, NFN-76 or MOM-181-4-F (E).

Exemplary CD3 binding agents (cd3γ/δ/epsilon, cd3γ, cd3δ, cd3γ/epsilon, cd3δ/epsilon, or cd3epsilon) may be antibodies selected from the group consisting of: MEM-57 (CD 3. Gamma./delta./epsilon., enzoLife Sciences), MAB100 (CD 3. Epsilon., R)&D Systems), CD3-H5 (CD 3 epsilon, abnova Corporation), CD3-12 (CD 3 epsilon, cell Signaling Technology), LE-CD3 (CD 3 epsilon, santa Cruz Biotechnology, inc.), NBP1-31250 (CD 3 gamma, novus Biologicals), 16669-1-AP (CD 3 delta, invitrogen), and antigen binding fragments thereof. In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H Domain and V _L Domain: MEM-57 (CD 3. Gamma./delta./epsilon., enzoLife Sciences), MAB100 (CD 3. Epsilon., R)&D Systems)、CD3-H5(CD3ε，Abnova Corporation)、CD3-12 (CD 3 ε, cell Signaling Technology), LE-CD3 (CD 3 ε, santa Cruz Biotechnology, inc.), NBP1-31250 (CD 3 γ, novus Biologicals) and 16669-1-AP (CD 3 δ, invitrogen). In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H And V _L Heavy chain CDRs of a sequence ₁ 、CDR ₂ And CDR ₃ Light chain CDRs ₁ 、CDR ₂ And CDR ₃ It is determined according to Kabat (see, kabat et al, (1991) Sequences of Proteins of Immunological Interest, NIH Publication No.91-3242, bethesda), chothia (see, e.g., chothia C and Lesk A M, (1987), J.MOL.BIOL.196:901-917), macCalum (see, macCalum R M et al, (1996) J.MOL.BIOL.262:732-745), or any other CDR determining method known in the art: MEM-57 (CD 3. Gamma./delta./epsilon., enzoLife Sciences), MAB100 (CD 3. Epsilon., R)&D Systems), CD3-H5 (CD 3 ε, abnova Corporation), CD3-12 (CD 3 ε, cell Signaling Technology), LE-CD3 (CD 3 ε, santa Cruz Biotechnology, inc.), NBP1-31250 (CD 3 γ, novus Biologicals), and 16669-1-AP (CD 3 δ, invitrogen).

Exemplary CD3 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following clones: hsp34, OKT-3, UCHT1, 38.1, HIT3a, RFT8, SK7, BC3, SP34-2, HU291, TRX4, cetuximab (Catumaxomab), tilizumab (teplizumab), 3-106, 3-114, 3-148, 3-190, 3-271, 3-550, 4-10, 4-48, H2C, F12Q, I2C, SP, 3F3A1, CD3-12, 301, RIV9, JB38-29, JE17-74, GT0013, 4E2, 7A4, 4D10A6, SPV-T3b, M2AB, ICO-90, 30A1 or Hu38E4.v1 (U.S. patent application 20200299409A 1), REGN 58 (U.S. patent application 20200024356A 1), bowmatrix https://go.drugbank.com/drugs/DB09052/polypeptide_ sequences.fasta). In some embodiments, the conjugate comprises a Fab, wherein the Fab comprises (a) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 2 or 3.

Exemplary CD4 binding agents may be antibodies selected from the group consisting of: abaruzumab (https:// www.genome.jp/dbget-bin/www_bgetD 09575), AF1856 (R)&D Systems)、MAB554(R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation), CAL4 (Abcam) and antigen binding fragments thereof. In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H Domain and V _L Domain: AF1856 (R)&D Systems)、MAB554(R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation), and CAL4 (Abcam). In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H And V _L Heavy chain CDRs of a sequence ₁ 、CDR ₂ And CDR ₃ Light chain CDRs ₁ 、CDR ₂ And CDR ₃ It is determined according to Kabat (see, kabat et al, (1991) Sequences of Proteins of Immunological Interest, NIH Publication No.91-3242, bethesda), chothia (see, e.g., chothia C and Lesk A M, (1987), J.MOL.BIOL.196:901-917), macCalum (see, macCalum R M et al, (1996) J.MOL.BIOL.262:732-745), or any other CDR determining method known in the art: AF1856 (R) &D Systems)、MAB554(R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation), and CAL4 (Abcam).

Exemplary CD4 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following clones: abamelizumab, OKT4, RPA-T4, S3.5, SK3, N1UG0, RIV6, OTI18E3, MEM-241, B486A1, RFT-4g, 7E14, MDX.2, MEM-115, MEM-16, ICO-86, edu-2 or Abamelizumab (ilbalizumab).

Exemplary CD5 binding agents may be antibodies selected from the group consisting of: he3, MAB1636 (R&D Systems)、AF1636(R&D Systems)、MAB115(R&D Systems), C5/473+CD5/54/F6 (Abcam), CD5/54/F6 (Abcam), 65152 (Proteintech) and antigen-binding fragments thereof. In some embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H Domain and V _L Domain: MAB1636 (R)&D Systems)、AF1636(R&D Systems)、MAB115(R&D Systems), C5/473+CD5/54/F6 (Abcam), CD5/54/F6 (Abcam), and 65152 (Proteintech). In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H And V _L Heavy chain CDRs of a sequence ₁ 、CDR ₂ And CDR ₃ Light chain CDRs ₁ 、CDR ₂ And CDR ₃ It is determined according to Kabat (see, kabat et al, (1991) Sequences of Proteins of Immunological Interest, NIH Publication No.91-3242, bethesda), chothia (see, e.g., chothia C and Lesk A M, (1987), J.MOL.BIOL.196:901-917), macCalum (see, macCalum R M et al, (1996) J.MOL.BIOL.262:732-745), or any other CDR determining method known in the art: MAB1636 (R) &D Systems)、AF1636(R&D Systems)、MAB115(R&D Systems), C5/473+CD5/54/F6 (Abcam), CD5/54/F6 (Abcam), and 65152 (Proteintech).

Exemplary CD5 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following clones: zolimumab (zolimomab), 5D7, L17F12, and UCHT2, 1D8, 3I21, 4H10, 8J23, 5O4, 4H2, 5G2, 8G8, 6M4, 2E3, 4E24, 4F10, 7J9, 7P9, 8E24, 6L18, 7H7, 1E7, 8J21, 7I11, 8M9, 1P21, 2H11, 3M22, 5M6, 5H8, 7I19, 1A2, 8E15, 8C10, 3P16, 4F3, 5M24, 5O24, 7B16, 1E8, 2H16, BLa1, 1804, DK23, cris1, MEM-32, H65, 4C7, OX-19, leu-1, 53-7.3, 4H8E6, T101, EP-52, D-9, 3, N-20, 3C 10, DK-35S-35, 3/C35, 3M-35S-35, 3/C35 (MOS-35/C35) or MOS-35 (MOF 35/S).

Exemplary CD7 binding agents may be antibodies selected from the group consisting of: MAB7579 (R)&D Systems)、AF7579(R&D Systems), EPR22065 (Abcam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals), NBP2-38440 (Novus Biologicals) and antigen binding fragments thereof. In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H Domain and V _L Domain: MAB7579 (R)&D Systems)、AF7579(R&D Systems), EPR22065 (Abcam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals) and NBP2-38440 (Novus Biologicals). In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H And V _L Heavy chain CDRs of a sequence ₁ 、CDR ₂ And CDR ₃ Light chain CDRs ₁ 、CDR ₂ And CDR ₃ According to Kabat (see, kabat et al, (1991) Sequences of Proteins)of Immunological Interest, NIH Publication No.91-3242, bethesda), chothia (see, e.g., chothia C and Lesk A M, (1987), J.MOL.BIOL.196:901-917), macCalum (see, macCallum R M et al, (1996), J.MOL.BIOL.262:732-745), or any other CDR determination method known in the art: MAB7579 (R)&D Systems)、AF7579(R&D Systems), EPR22065 (Abcam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals) and NBP2-38440 (Novus Biologicals).

Exemplary CD7 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following clones: TH-69, 3Afl1, T3-3A1, 124-1D1, 3A1f, CD7-6B7 or VHH6.

Exemplary CD8 (CD 8 a, CD8 a/β or CD8 β) binding agents may be antibodies selected from the group consisting of: 2.43 (Invitrogen), du CD8-1 (CD 8. Alpha., invitrogen), 9358-CD (CD 8. Alpha./beta., R)&D System s)、MAB116(CD8α，R&D Systems), ab4055 (CD 8. Alpha., abcam), C8/144B (CD 8. Alpha., novus Biologicals), YTS105.18 (CD 8. Alpha., novus Biologicals), TRX2 (https:// patents. Ju still. Com/patent/20170198045), and antigen binding fragments thereof. In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H Domain and V _L Domain: 2.43 (Invitrogen), 51.1 (ATCC HB-230), du CD8-1 (CD 8. Alpha., invitrogen), 9358-CD (CD 8. Alpha./beta., R)&D Systems)、MAB116(CD8α，R&D Systems), ab4055 (CD 8a, abcam), C8/144B (CD 8a, novus Biologicals) and Y TS105.18 (CD 8a, novus Biologicals). In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H And V _L Heavy chain CDRs of a sequence ₁ 、CDR ₂ And CDR ₃ Light chain CDRs ₁ 、CDR ₂ And CDR ₃ It is determined according to Kabat (see, kabat et al, (1991) Sequences of Proteins of Immunological Interest, NIH Publication No.91-3242, bethesda), chothia (see, e.g., chothia C and Lesk A M, (1987), J.MOL.BIOL.196:901-917), macCalum (see, macCalum R M et al, (1996) J.MOL.BIOL.262:732-745), or any other CDR determining method known in the art: 2.43 (In vitro gen), du CD8-1 (CD 8. Alpha., invitrogen), 9358-CD (CD 8. Alpha./beta., R)&D Systems)、M AB116(CD8α，R&D Systems), ab4055 (CD 8a, abcam), C8/144B (CD 8a, novus Biologicals) and YTS105.18 (CD 8a, novus Biologicals).

Exemplary CD8 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following clones: OKT-8, 51.1, S6F1, TRX2, and UCHT4, SP16, 3B5, C8-144B, HIT a, RAVB3, LT8, 17D8, MEM-31, MEM-87, RIV11, DK-25, YTC141.1HL, or YTC182.20. In some embodiments, the conjugate comprises a Fab, wherein the Fab comprises a heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 7.

Exemplary CD137 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following clones: 4B4-1, P566 or Urelumab (Urelumab). Exemplary CD28 binding agents may be selected from antibodies or antibody fragments employing the CDRs of clone TAB 08. Exemplary CD45 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following clones: BC8, 9.4, 4B2, tu116 or GAP8.3. Exemplary CD18 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following clones: 1B4, TS1/18, MEM-48, YFC118-3, TA-4, MEM-148 or R3-3,24. Exemplary CD11a binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following clones: MHM24 or Efalizumab (Efalizumab). Exemplary IL-2 receptor binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following clones: YTH 906.9HL, IL2R.1, BC96, B-B10, 216, MEM-181, ITYV, MEM-140, ICO-105, daclizumab (Daclizumab), or selected from IL2 or an IL2 fragment. Exemplary IL-15R binders may be selected from antibodies or antibody fragments employing the CDRs of the following clones: JM7A4 or OTI3D5, or selected from IL15 or IL15 fragment. Exemplary TLR2 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following clones: JM22-41, TL2.1, 11G7 or TLR2.45. Exemplary TLR4 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following clones: HTA125 or 76B357-1. Exemplary TLR5 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following clones: 85B152-5 or 9D759-2. Exemplary GL7 binding agents may be selected from antibodies or antibody fragments employing CDRs of clone GL 7.

Exemplary PD1 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following clones: MIH4, J116, J150, OTIB11, OTI17B10, OTI3A1, or OTI16D4. In addition, exemplary anti-PD-1 antibodies are described, for example, in U.S. patent nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (nivolumab)Bristol-Myers Squibb Co.), pembrolizumab (pembrolizumab) (-A.sub.f.)>Merck Sharp&Dohme corp.), PDR001 (Novartis Pharmaceuticals), and pidirizumab (CT-011, cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. patent nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (atezolizumab) (-)>Genentech), devalumab (durvalumab) (AstraZeneca), MEDI4736, avistuzumab (avelumab), and BMS 936559 (Bristol Myers Squibb co.).

Exemplary CTLA-4 binding agents can be selected from antibodies or antibody fragments employing the CDRs of the following clones: ER4.7G.11[7G11], OTI9G4, OTI9F3, OTI3A5, A3.4H2.H12, 14D3, OTI3A12, OTI1A11, OTI1E8, OTI3B11, OTI3D2, OTI10C8, OTI2E9, OTI6F1, OTI7D3, OTI85B, OTI C6. Exemplary anti-CTLA-4 antibodies are described in U.S. patent nos. 6,984,720, 6,682,736, 7,311,910, 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815 and 8,883,984; international (PCT) publication Nos. WO 98/42752, WO 00/37504 and WO 01/14424; in european patent No. EP 1212422 B1. Exemplary CTLA-4 antibodies include ipilimumab (ipilimumab) or tremelimumab (tremelimumab).

Exemplary tcrp binding agents may be antibodies selected from the group consisting of: h57-597 (Invitrogen), 8A3 (Novus Biologicals), R73 (TCRα/β, abcam), E6Z3S (TRBC 1/TCRβ, cell Signaling Technology) and antigen binding fragments thereof. In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H Domain and V _L Domain: h57-597 (Invitrogen), 8A3 (Novus Biologicals), R73 (TCRα/β, abcam), and E6Z3S (TRBC 1/TCRβ, cell Signaling Technology). In certain embodiments, the binding agent comprises V of an antibody selected from the group consisting of _H And V _L Heavy chain CDRs of a sequence ₁ 、CDR ₂ And CDR ₃ Light chain CDRs ₁ 、CDR ₂ And CDR ₃ It is determined according to Kabat (see, kabat et al, (1991) Sequences of Proteins of Immunological Interest, NIH Publication No.91-3242, bethesda), chothia (see, e.g., chothia C and Lesk A M, (1987), J.MOL.BIOL.196:901-917), macCalum (see, macCalum R M et al, (1996) J.MOL.BIOL.262:732-745), or any other CDR determining method known in the art: h57-597 (Invitrogen), 8A3 (Novus Biologicals), R73 (TCRα/β, abcam), and E6Z3S (TRBC 1/TCRβ, cell Signaling Technology).

Exemplary CD137 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following clones: 4B4-1, P566 or Urelumab (Urelumab).

In some embodiments, the immune cell targeting group comprises an antibody selected from the group consisting of: fab, F (ab ') 2, fab' -SH, fv and scFv fragments. In some embodiments, the antibody is a human or humanized antibody. In some embodiments, the immune cell targeting group comprises a Fab or immunoglobulin single variable domain, such as a nanobody. In some embodiments, the immune cell targeting group comprises a Fab that does not contain native interchain disulfide bonds. For example, in some embodiments, the Fab comprises a heavy chain fragment comprising a C233S substitution and/or a light chain fragment comprising a C214S substitution, numbered according to Kabat. In some embodiments, the immune cell targeting group comprises a Fab containing one or more unnatural interchain disulfide bonds. In some embodiments, the interchain disulfide bond is between two unnatural cysteine residues on the light chain fragment and the heavy chain fragment, respectively. For example, in some embodiments, the Fab comprises a heavy chain fragment comprising an F174C substitution and/or a light chain fragment comprising an S176C substitution, numbered according to Kabat. In some embodiments, the Fab comprises a heavy chain fragment comprising F174C and C233S substitutions and/or a light chain fragment comprising S176C and C214S substitutions, numbered according to Kabat. In some embodiments, the immune cell targeting group comprises a C-terminal cysteine residue. In some embodiments, the immune cell targeting group comprises a Fab containing a cysteine at the C-terminus of the heavy or light chain fragment. In some embodiments, the Fab further comprises one or more amino acids between the heavy chain of the Fab and the C-terminal cysteine. For example, in some embodiments, the Fab comprises two or more amino acids derived from an antibody hinge region (e.g., a partial hinge sequence) between the C-terminus and the C-terminal cysteine of the Fab. In some embodiments, the Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and wherein the immune cell targeting group further comprises a single chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain by an amino acid linker. In some embodiments, the Fab antibody is a DS Fab, noDS Fab, bDS Fab-ScFv, as shown in fig. 47.

In some embodiments, the immune cell targeting group comprises an immunoglobulin single variable domain, such as a nanobody (e.g., V _HH ). In some embodiments, the nanobody comprises a cysteine at the C-terminus. In some embodiments, the nanobody further comprises a spacer comprised in the V _HH One or more amino acids between the domain and the C-terminal cysteine. In some embodiments, the spacer comprises one or more glycine residues, e.gTwo glycine residues. In some embodiments, the immune cell targeting group comprises two or more V _HH A domain. In some embodiments, the two or more V _HH The domains are linked by amino acid linkers. In some embodiments, the amino acid linker comprises one or more glycine and/or serine residues (e.g., one or more repeats of the sequence GGGGS). In some embodiments, the immune cell targeting group comprises a first V linked to an antibody CH1 domain _HH Domain and second V linked to antibody light chain constant domain _HH A domain, and wherein the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds (e.g., interchain disulfide bonds). In some embodiments, the immune cell targeting group comprises V linked to an antibody CH1 domain _HH A domain, and wherein the antibody CH1 domain is linked to an antibody light chain constant domain by one or more disulfide bonds. In some embodiments, the CH1 domain comprises F174C and C233S substitutions and the light chain constant domain comprises S176C and C214S substitutions, numbered according to Kabat. In some embodiments, the antibody is an ScFv, V _HH 、2xV _HH 、V _HH -CH 1/empty Vk or V _HH 1-CH1/V _HH -2-Nb bDS, as shown in fig. 47.

Exemplary targeting moieties can have amino sequences as set forth below:

anti-CD 3 hSP34-Fab sequence:

hSP34 Heavy Chain (HC) sequence (SEQ ID NO: 1):

EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

hSP34-mlam Light Chain (LC) sequence (mouse lambda) (SEQ ID NO: 2):

QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADSS

SP34-hlam LC (human lambda) (SEQ ID NO: 3):

QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAESS

anti-CD 3 Hu291-Fab sequence:

Hu291 HC(SEQ ID NO:4)：

QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

Hu291 LC(SEQ ID NO:5)：

MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 8 TRX2-Fab sequence:

TRX2 HC(SEQ ID NO:6)：

QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

TRX2 LC(SEQ ID NO:7)：

DIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 8 OKT8-Fab sequence:

OKT8 HC(SEQ ID NO:8)：

QVQLVQSGAEDKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNTLYASKFQGRVTITADTSSNTAYMELSSLRSEDTAVYYCGRGYGYYVFDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

OKT8 LC(SEQ ID NO:9)：

DIVMTQSPSSLSASVGDRVTITCRTSRSISQYLAWYQEKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 4 ibalizumab-Fab sequence:

ibalizumab HC (SEQ ID NO: 10):

QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

abamectin LC (SEQ ID NO: 11):

DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 5 He3-Fab sequence:

He3 HC(SEQ ID NO:12)：

EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

He3 LC(SEQ ID NO:13)：

DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 7 TH-69-Fab sequence:

TH-69HC(SEQ ID NO:14)：

EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

TH-69LC(SEQ ID NO:15)：

DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

anti-CD 2 TS2/18.1-Fab sequence:

TS2/18.1HC(SEQ ID NO:16)：

EVQLVESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

TS2/18.1LC(SEQ ID NO:17)：

DIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYFCQNGHNFPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 2 9.6-Fab sequence:

9.6HC(SEQ ID NO:18)：

QVQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

9.6LC(SEQ ID NO:19)：

NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 2 9-1-Fab sequence:

9-1HC(SEQ ID NO:20)：

QVQLQQPGTELVRPGSSVKLSCKASGYTFTSYWVNWVKQRPDQGLEWIGRIDPYDSETHYNQKFTDKAISTIDTSSNTAYMQLSTLTSDASAVYYCSRSPRDSSTNLADWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

9-1LC(SEQ ID NO:21)：

DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

mutOKT8-Fab sequence:

mutOKT8 HC(SEQ ID NO:22)：

QVQLVQSGAEDKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNTLYASKFQGRVTITADTSSNTAYMELSSLRSEDTAVYYCGRGAGAYVFDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

mutOKT8 LC(SEQ ID NO:23)：

DIVMTQSPSSLSASVGDRVTITCRTSRSISAALAWYQEKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES。

in certain embodiments, the targeting group or immune cell targeting group (e.g., T cell targeting agent, B cell targeting agent, or NK cell targeting agent) can be covalently coupled to the lipid via a polyethylene glycol (PEG) -containing linker.

In other embodiments, the lipid used to produce the conjugate may be selected from distearoyl-phosphatidylethanolamine (DSPE):

dipalmitoyl-phosphatidylethanolamine (DPPE):

dimyristoyl-phosphatidylethanolamine (DMPE):

distearoyl-glycerol-phosphate glycerol (DSPG):

dimyristoyl-glycerol (DMG):

distearoyl glycerol (DSG):

and

n-palmitoyl-sphingosine (C16-ceramide)

The immune cell targeting group can be covalently coupled to the lipid directly or via a linker (e.g., a linker containing polyethylene glycol (PEG)). In certain embodiments, the PEG is PEG 1000, PEG 2000, PEG 3400, PEG 3000, PEG 3450, PEG 4000, or PEG 5000. In certain embodiments, the PEG is PEG 2000.

In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in a range of 0.001-0.5 mole percent, 0.001-0.3 mole percent, 0.002-0.2 mole percent, 0.01-0.1 mole percent, 0.1-0.3 mole percent, or 0.1-0.2 mole percent.

In certain embodiments, the lipid-immune cell targeting agent conjugate comprises DSPE, a PEG component, and a targeting antibody. In certain embodiments, the antibody is a T cell targeting agent, such as an anti-CD 2 antibody, an anti-CD 3 antibody, an anti-CD 4 antibody, an anti-CD 5 antibody, an anti-CD 7 antibody, an anti-CD 8 antibody, or an anti-TCR β antibody.

Exemplary lipid-immune cell targeting group conjugates include DSPE and PEG 2000, e.g., as described in Nellis et al (2005) biotechnol. Prog.21, 205-220. Exemplary conjugates comprise a structure of formula V, wherein the scFv represents an engineered antibody binding site that binds to a target of interest. In certain embodiments, the engineered antibody binding site binds to any of the targets described above. In certain embodiments, the engineered antibody binding site may be, for example, an engineered anti-CD 3 antibody or an engineered anti-CD 8 antibody. In certain embodiments, the engineered antibody binding site may be, for example, an engineered anti-CD 2 antibody or an engineered anti-CD 7 antibody.

Examples of compounds of formula (V) are shown below:

it is contemplated that the scFv in formula V may be replaced with an intact antibody or an antigenic fragment thereof (e.g., fab).

Another example of a compound of formula (VI) is shown below:

its production is described in Nellis et al (2005) supra or in U.S. Pat. No. 7,022,336. It is contemplated that the Fab of formula VI can be conjugated to an intact antibody or antigen fragment thereof (e.g., (Fab') ₂ Fragments) or engineered antibody binding sites (e.g., scFv).

Other lipid-immune cell targeting group conjugates are described, for example, in U.S. patent No. 7,022,336, wherein the targeting group can be replaced with a targeting group of interest (e.g., a targeting group that binds to a T cell or NK cell surface antigen as described above).

In certain embodiments, the lipid component of the exemplary conjugates of formula IV may be based on ionizable cationic lipids described herein, such as ionizable cationic lipids of formula I, formula II, or formula III. For example, exemplary ionizable cationic lipids may be selected from:

or a salt thereof.

In certain embodiments, exemplary ionizable cationic lipids can be compounds of the formula:

or a salt thereof.

In some embodiments, an exemplary ionizable cationic lipid can be a compound of the formula:

or a salt thereof.

In other embodiments, exemplary ionizable cationic lipids can be compounds of the formula:

or a salt thereof.

Or a salt thereof.

In certain embodiments, the lipid-based conjugate of formula III may comprise:

wherein scFv represents an engineered antibody binding site that binds to a target described above (e.g., CD2, CD3, CD7, or CD 8).

In certain embodiments, the lipid blend may further comprise free PEG-lipids in order to reduce the amount of non-specific binding via the targeting group. The free PEG-lipid may be the same as or different from the PEG-lipid included in the conjugate. In certain embodiments, the free PEG-lipid is selected from PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), N- (methylpolyoxyoxycarbonyl) -1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1, 2-dimyristoyl-rac-glycero-3-methylpolyethylene oxide (PEG-DMG), 1, 2-dipalmitoyl-rac-glycero-3-methylpolyethylene oxide (PEG-dpp), 1, 2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG), 1, 2-distearoyl-rac-glycero-3-methylpolyethylene oxide (PEG-DSG), N-palmitoyl-sphingosine-1- { succinyl [ methoxy (polyethylene glycol) ] (PEG-ceramide), DSPE-PEG-cysteine, or derivatives thereof, all having an average PEG length of between 2000-5000, 3400, or 5000. The final composition may contain a mixture of two or more of these pegylated lipids. In certain embodiments, the LNP composition comprises a mixture of PEG-lipids having myristoyl and stearoyl chains.

In certain embodiments, the derivative of PEG-lipid has a hydroxyl or carboxylic acid end group at the PEG terminus.

The lipid-immune cell targeting group conjugates can be incorporated into LNPs as described below, for example into LNPs containing, for example, ionizable cationic lipids, sterols, neutral phospholipids, and PEG-lipids. It is contemplated that in certain embodiments, the LNP containing the lipid-immune cell targeting group may contain an ionizable cationic lipid as described herein, or a cationic lipid as described in, for example, U.S. patent No. 10,221,127, 10,653,780 or U.S. published application No. US2018/0085474, US2016/0317676, international publication No. WO2009/086558, or Miao et al (2019) NATURE BIOTECH 37:1174-1185, or Jayaraman et al (2012) ANGEW CHEM int.51:8529-8533. In other embodiments, the cationic lipid may be selected from the ionizable cationic lipids set forth in table 1.

Table 1.

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The LNP can be formulated using the methods and other components described in the following sections.

Lipid nanoparticle compositions

The present invention provides Lipid Nanoparticle (LNP) compositions comprising a lipid blend comprising an ionizable cationic lipid described herein and/or a lipid-immune cell targeting agent conjugate described herein. In certain embodiments, the lipid blend may comprise an ionizable cationic lipid as described herein and one or more of a sterol, a neutral phospholipid, a PEG-lipid, and a lipid-immune cell targeting group conjugate.

In certain embodiments, the ionizable cationic lipids described herein can be present in the lipid blend in a range of 30-70 mole percent, 30-60 mole percent, 30-50 mole percent, 40-70 mole percent, 40-60 mole percent, 40-50 mole percent, 50-70 mole percent, 50-60 mole percent, or about 30 mole percent, about 35 mole percent, about 40 mole percent, about 45 mole percent, about 50 mole percent, about 55 mole percent, about 60 mole percent, about 65 mole percent, or about 70 mole percent.

Sterols

In certain embodiments, the lipid blend of lipid nanoparticles may comprise a sterol component, such as one or more sterols selected from the group consisting of: cholesterol, fucosterol, beta-sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, and brassinosteroids. In certain embodiments, the sterol is cholesterol.

The sterols (e.g., cholesterol) may be present in the lipid blend in a range of 20-70 mole percent, 20-60 mole percent, 20-50 mole percent, 30-70 mole percent, 30-60 mole percent, 30-50 mole percent, 40-70 mole percent, 40-60 mole percent, 40-50 mole percent, 50-70 mole percent, 50-60 mole percent, or about 20 mole percent, about 25 mole percent, about 30 mole percent, about 35 mole percent, about 40 mole percent, about 45 mole percent, about 50 mole percent, about 55 mole percent, about 60 mole percent, or about 65 mole percent.

Neutral phospholipids

In certain embodiments, the lipid blend of the lipid nanoparticle may comprise one or more neutral phospholipids. The neutral phospholipid may be selected from phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM).

Other neutral phospholipids may be selected from distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycerophosphoryl choline (DSPC), dioleoyl-glycerophosphoryl ethanolamine (DOPE), dioleoyl-glycerophosphoryl choline (DLPC), dimyristoyl-glycerophosphoryl choline (DMPC), dioleoyl-glycerophosphoryl choline (DOPC), dipalmitoyl-glycerophosphoryl choline (DPPC), heneicosyl-glycerophosphoryl choline (DUPC), palmitoyl-oleoyl-glycerophosphoryl choline (POPC), octadienyl-glycerophosphoryl choline, oleoyl-cholesterol hemisuccinyl-glycerophosphoryl choline, hexadecyl-glycerophosphoryl choline, dioleoyl-glycerophosphoryl choline, ditetraenoyl-glycero-3-phosphocholine, docosahexaenoyl-glycerophosphoryl choline or sphingomyelin.

The neutral phospholipid may be present in the lipid blend in a range of 1-10 mole percent, 1-15 mole percent, 1-12 mole percent, 1-10 mole percent, 3-15 mole percent, 3-12 mole percent, 3-10 mole percent, 4-15 mole percent, 4-12 mole percent, 4-10 mole percent, 4-8 mole percent, 5-15 mole percent, 5-12 mole percent, 5-10 mole percent, 6-15 mole percent, 6-12 mole percent, 6-10 mole percent, or about 1 mole percent, about 2 mole percent, about 3 mole percent, about 4 mole percent, about 5 mole percent, about 6 mole percent, about 7 mole percent, about 8 mole percent, about 9 mole percent, about 10 mole percent, about 11 mole percent, about 12 mole percent, about 13 mole percent, about 14 mole percent, or about 15 mole percent.

PEG-lipids

The lipid blend of lipid nanoparticles may include one or more PEG or PEG-modified lipids. Such species may alternatively be referred to as pegylated lipids. PEG lipids are lipids modified with polyethylene glycol. As noted above, when lipid-immune cell targeting groups are included in the lipid blend, free PEG-lipids may be included in the lipid blend to reduce or eliminate non-specific binding via the targeting groups.

The PEG lipids may be selected from the non-limiting group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, the PEG lipid may be PEG-dioleoyl glycerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-dppg), PEG-diiodoyl-glycerol-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoyl glycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycerol-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycerol-phosphoethanolamine (PEG-DOPE), 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide, or PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

In certain embodiments, the blend may contain free PEG-lipids, which may be selected from PEG-distearoyl glycerol (PEG-DSG), PEG-diacylglycerols (PEG-DAG, e.g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE), and PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE). In some embodiments, the free PEG-lipid comprises a diacyl phosphatidylcholine comprising a dipalmitoyl (C16) chain or a distearoyl (C18) chain.

The PEG-lipid may be present in the lipid blend in a range of 1-10 mole percent, 1-8 mole percent, 1-7 mole percent, 1-6 mole percent, 1-5 mole percent, 1-4 mole percent, 1-3 mole percent, 2-8 mole percent, 2-7 mole percent, 2-6 mole percent, 2-5 mole percent, 2-4 mole percent, 2-3 mole percent, or about 1 mole percent, about 2 mole percent, about 3 mole percent, about 4 mole percent, or about 5 mole percent. In some embodiments, the PEG-lipid is a free PEG-lipid.

In some embodiments, the PEG-lipid may be present in the range of 0.01-10 mole percent, 0.01-5 mole percent, 0.01-4 mole percent, 0.01-3 mole percent, 0.01-2 mole percent, 0.01-1 mole percent, 0.1-10 mole percent, 0.1-5 mole percent, 0.1-4 mole percent, 0.1-3 mole percent, 0.1-2 mole percent, 0.1-1 mole percent, 0.5-10 mole percent, 0.5-5 mole percent, 0.5-4 mole percent, 0.5-3 mole percent, 0.5-2 mole percent, 0.5-1 mole percent, 1-2 mole percent, 3-4 mole percent, 4-5 mole percent, 5-6 mole percent, or 1.25-1.75 mole percent of the lipid. In some embodiments, the PET-lipid may be about 0.5 mole percent, about 1 mole percent, about 1.5 mole percent, about 2 mole percent, about 2.5 mole percent, about 3 mole percent, about 3.5 mole percent, about 4 mole percent, about 4.5 mole percent, about 5 mole percent, or about 5.5 mole percent of the lipid blend. In some embodiments, the PEG-lipid is a free PEG-lipid.

In some embodiments, the lipid anchor length of the PEG-lipid is C14 (as in PEG-DMG). In some embodiments, the lipid anchor length of the PEG-lipid is C16 (as in DPG). In some embodiments, the lipid anchor length of the PEG-lipid is C18 (as in PEG-DSG). In some embodiments, the backbone or headgroup of the PEG-lipid is diacylglycerol or phosphoethanolamine. In some embodiments, the PEG-lipid is a free PEG-lipid.

The LNP of the present disclosure may comprise one or more free PEG-lipids not conjugated to an immune cell targeting group and PEG-lipids conjugated to an immune cell targeting group. In some embodiments, the free PEG-lipid comprises the same or different lipid as the lipid in the lipid-immune cell targeting group conjugate.

Immune cell targeting group conjugates

In certain embodiments, the lipid blend may further comprise a lipid-immune cell targeting group conjugate as described above in section III.

The lipid-immune cell targeting group conjugate may be present in the lipid blend in a range of 0.001-0.5 mole percent, 0.001-0.1 mole percent, 0.01-0.5 mole percent, 0.05-0.5 mole percent, 0.1-0.3 mole percent, 0.1-0.2 mole percent, 0.2-0.3 mole percent, about 0.01 mole percent, about 0.05 mole percent, about 0.1 mole percent, about 0.15 mole percent, about 0.2 mole percent, about 0.25 mole percent, about 0.3 mole percent, about 0.35 mole percent, about 0.4 mole percent, about 0.45 mole percent, or about 0.5 mole percent.

The LNP composition may further comprise a payload, such as the payload described below, in addition to the lipids present in the lipid blend. In certain embodiments, the payload is a nucleic acid, e.g., DNA or RNA, e.g., mRNA, transfer RNA (tRNA), microrna, or small interfering RNA (siRNA).

In certain embodiments, the number of nucleotides in the nucleic acid is from about 400 to about 6000.

Production of lipid nanoparticles

Typically, the LNP is produced by using rapid mixing via an orbital scroll or by microfluidic mixing. The orbital scroll mixing is accomplished by: the alcoholic solution of the lipid was added rapidly to the aqueous solution of the nucleic acid of interest, and then immediately vortexed at 2,500 rpm. Microfluidic mixing is achieved by mixing aqueous and organic streams in a microfluidic channel at controlled flow rates using, for example, a nanoAsssembrr device featuring optimized mixing chamber geometry and a microfluidic chip (Precision Nanosystems, vancouver, columbia, not shown).

In certain embodiments, the resulting LNP composition comprises a lipid blend containing, for example, from about 40 mole percent to about 60 mole percent of one or more ionizable cationic lipids described herein, from about 35 mole percent to about 50 mole percent of one or more sterols, from about 5 mole percent to about 15 mole percent of one or more neutral lipids, and from about 0.5 mole percent to about 5 mole percent of one or more PEG-lipids.

Physical Properties of lipid nanoparticles

The characteristics of the LNP composition may depend on the components contained in the Lipid Nanoparticle (LNP) composition, absolute or relative amounts thereof. The characteristics may also vary depending on the method and conditions of preparation of the LNP composition.

The LNP composition can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the LNP composition. Dynamic light scattering or potentiometry (e.g., potentiometry) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as Zetasizer Nano ZS (Malvern Instruments Ltd, UK Wright Markov) may also be used to measure various characteristics of the LNP composition, such as particle size, polydispersity index, and zeta potential. RNA encapsulation efficiency was determined by a combination of methods that rely on RNA binding dyes (ribogreen, cybergreen for determining the fraction of dye accessible RNA) and LNP de-formulation, followed by HPLC analysis of total RNA content.

In some embodiments, the LNP has an average diameter in the range of 1-250nm, 1-200nm, 1-150nm, 1-100nm, 50-250nm, 50-200nm, 50-150nm, 50-100nm, 75-250nm, 75-200nm, 75-150nm, 75-100nm, 100-250nm, 100-200nm, 100-150 nm. In certain embodiments, the LNP composition can have an average diameter of about 1nm, about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 110nm, about 120nm, about 130nm, about 140nm, about 150nm, about 160nm, about 170nm, about 180nm, about 190nm, or about 200 nm. In some embodiments, the LNP has an average diameter of about 100 nm.

Alternatively or additionally, the LNP composition can have a polydispersity index in a range from 0.05-1, 0.05-0.75, 0.05-0.5, 0.05-0.4, 0.05-0.3, 0.05-0.2, 0.08-1, 0.08-0.75, 0.08-0.5, 0.08-0.4, 0.08-0.3, 0.08-0.2, 0.1-1, 0.1-0.75, 0.1-0.5, 0.1-0.4, 0.1-0.3, 0.1-0.2. In certain embodiments, the polydispersity index is in the range of 0.1 to 0.25, 0.1 to 0.2, 0.1 to 0.19, 0.1 to 0.18, 0.1 to 0.17, 0.1 to 0.16, or 0.1 to 0.15.

Alternatively or additionally, the LNP composition may have a zeta potential of about-30 mV to about +30 mV. In certain embodiments, the LNP composition has a zeta potential of about-10 mV to about +20 mV. The zeta potential may vary with the pH. Thus, in certain embodiments, the LNP composition may have a zeta potential of about-10 mV to about +30mV or about 0mV to +30mV or about +5mV to about +30mV at pH 5.5 or pH 5, and/or may have a zeta potential of about-30 mV to about +5mV or about-20 mV to about +15mV at pH 7.4.

V. payload

The LNP composition can comprise an agent, such as a nucleic acid molecule, for delivery to a cell (e.g., an immune cell) or tissue (e.g., a cell (e.g., an immune cell) or tissue of a subject).

The LNP composition of the invention can include a nucleic acid, e.g., DNA or RNA, such as mRNA, tRNA, microRNA, siRNA, or dicer substrate siRNA. It is contemplated that the nucleic acid may contain naturally occurring components, such as naturally occurring bases, sugars, or linkages (e.g., phosphodiester linkages); or may contain non-naturally occurring components or modifications (e.g., thioester linkages). For example, the nucleic acid may be synthesized to contain base, sugar, linker modifications known to those skilled in the art. Furthermore, the nucleic acid may be linear or circular, or have any desired configuration. The LNP composition can include a plurality of nucleic acid molecules (e.g., a plurality of RNA molecules), which can be the same or different.

In certain embodiments, the payload is mRNA. In certain embodiments, a particular LNP composition may contain a number of mRNA molecules, which may be the same or different. In certain embodiments, one or more LNP compositions comprising one or more different mrnas can be combined with and/or contacted simultaneously with a cell. It is contemplated that the mRNA may include one or more of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and/or a 5' cap structure. The mRNA may encode a receptor, such as a Chimeric Antigen Receptor (CAR), for use in, for example, an immune disorder, an inflammatory disorder, or cancer. In addition, the mRNA may encode an antigen for use in a therapeutic or prophylactic vaccine, for example, for treating or preventing a pathogen (e.g., a microbial or viral pathogen) infection, or for reducing or ameliorating side effects caused directly or indirectly by such an infection.

In certain embodiments, the LNP composition can include one or more other components including, but not limited to, one or more pharmaceutically acceptable excipients, hydrophobic small molecules, therapeutic agents, carbohydrates, polymers, permeability enhancing molecules, and surface modifying agents.

In some embodiments, the wt/wt ratio of the lipid component to the payload (e.g., mRNA) in the resulting LNP composition is from about 1:1 to about 50:1. In certain embodiments, the wt/wt ratio of the lipid component to the payload (e.g., mRNA) in the resulting composition is from about 5:1 to about 50:1. In certain embodiments, the wt/wt ratio is from about 5:1 to about 40:1. In certain embodiments, the wt/wt ratio is from about 10:1 to about 40:1. In certain embodiments, the wt/wt ratio is from about 15:1 to about 25:1.

In certain embodiments, the encapsulation efficiency of the payload (e.g., mRNA) in the lipid nanoparticle is at least 50%. In certain embodiments, the encapsulation efficiency is at least 80%, at least 90%, or greater than 90%.

RNA payload

In certain embodiments, the RNA payload is an mRNA, tRNA, microrna, or siRNA payload.

In certain embodiments, the lipid nanoparticle composition is optimized for delivery of RNA (e.g., mRNA) to a target cell for translation within the cell. The mRNA may be naturally or non-naturally occurring mRNA. An mRNA may include one or more modified nucleobases, nucleosides, or nucleotides.

The nucleobases may be selected from the non-limiting group consisting of: adenine, guanine, uracil, cytosine, 7-methylguanine, 5-methylcytosine, 5-hydroxymethylcytosine, thymine, pseudouracil, dihydrouracil, hypoxanthine and xanthine.

Nucleosides of mRNA are compounds that include a combination of a sugar molecule (e.g., a 5-carbon or 6-carbon sugar, such as pentose, ribose, arabinose, xylose, glucose, galactose, or deoxy derivatives thereof) with a nucleobase. The nucleoside can be a classical nucleoside (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine) or an analog thereof, and can include one or more substitutions or modifications.

The nucleotides of mRNA are compounds that contain a nucleoside and a phosphate group or alternative group (e.g., borophosphate, phosphorothioate, phosphoroselenate, phosphonate, alkyl, amidate, and glycerol). The nucleotide may be a classical nucleotide (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine monophosphate) or an analog thereof, and may include one or more substitutions or modifications including, but not limited to, alkyl, aryl, halo, oxo, hydroxy, alkoxy, and/or thio substitutions; one or more fused or open rings; oxidation of nucleobases, sugars and/or phosphoric acid or alternative components; and/or reduction. The nucleotide may include one or more phosphate or alternative groups. For example, a nucleotide may include a nucleoside and a triphosphate group. "nucleoside triphosphates" (e.g., guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, and uridine triphosphate) may refer to classical nucleoside triphosphates or analogs or derivatives thereof, and may include one or more substitutions or modifications as described herein.

The RNA can include a 5 'untranslated region, a 3' untranslated region, and/or a coding or translated sequence. mRNA can include any number of base pairs, including tens, hundreds, or thousands of base pairs. Any number (e.g., all, some, or none) of nucleobases, nucleosides, or nucleotides can be substituted, modified, or otherwise non-naturally occurring analogs of the classical species. In certain embodiments, all of a particular nucleobase type may be modified. For example, all cytosines in an mRNA may be 5-methylcytosine.

In certain embodiments, the mRNA may include a 5' cap structure, a chain termination nucleotide, a stem loop, a polyA sequence, and/or a polyadenylation signal.

The cap structure or cap class is a compound comprising two nucleoside moieties linked by a linker and may be selected from naturally occurring caps, non-naturally occurring caps or cap analogues. The cap species may include one or more modified nucleosides and/or linker moieties. For example, a natural mRNA cap may include a guanine nucleotide linked at its 5' position by a triphosphate bond and a guanine (G) nucleotide methylated at 7 position, e.g., m7G (5 ') ppp (5 ') G, typically written as m7GpppG. The cap species may also be an anti-reverse cap analogue. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m ' dGpppG, m7Gpppm7G, m ' dGpppG and m27 ' GppGppG.

Alternatively or additionally, the mRNA may include a chain terminating nucleoside. For example, chain terminating nucleosides can include those that are deoxy at the 2 'and/or 3' positions of their glycosyl groups. Such species may include 3' -deoxyadenosine (cordycepin), 3' -deoxyuridine, 3' -deoxycytosine, 3' -deoxyguanosine, 3' -deoxythymine, and 2',3' -dideoxynucleosides, such as 2',3' -dideoxyadenosine, 2',3' -dideoxyuridine, 2',3' -dideoxycytosine, 2',3' -dideoxyguanosine, and 2',3' -dideoxythymine.

Alternatively or additionally, the mRNA may include a stem loop, such as a histone stem loop. The stem loop may comprise 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotide base pairs. For example, the stem loop may comprise 4, 5, 6, 7 or 8 nucleotide base pairs. The stem loop may be located in any region of the mRNA. For example, the stem loop may be located in the untranslated region (5 'untranslated region or 3' untranslated region), the coding region, or the polyA sequence or the tail, before or after.

Alternatively or additionally, the mRNA may include polyA sequences and/or polyadenylation signals. The polyA sequence may consist entirely or predominantly of adenine nucleotides or analogues or derivatives thereof. The polyA sequence may be a tail located near the 3' untranslated region of an mRNA.

The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell. In some embodiments, the mRNA may encode an antibody, enzyme, growth factor, hormone, cytokine, viral protein (e.g., viral capsid protein), antigen, vaccine, or receptor. In some embodiments, the mRNA can encode an engineered receptor (e.g., CAR) or an antigen for use in a therapeutic vaccine (e.g., a cancer vaccine) or a prophylactic vaccine (e.g., a vaccine for minimizing the risk or severity of infection by a microbial or viral pathogen). In some embodiments, the mRNA encodes a polypeptide capable of modulating an immune response in the immune cell. In some embodiments, the mRNA encodes a polypeptide that is capable of reprogramming the immune cell. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or Chimeric Antigen Receptor (CAR).

Lipid compositions may be designed for one or more specific applications or targets. For example, the LNP composition can be designed to deliver mRNA to a particular cell, tissue, organ or system of a mammalian body or group thereof (e.g., the renal system). The physicochemical properties of the LNP composition can be altered so as to increase selectivity for a particular target site within the subject. For example, the granularity may be adjusted based on the fenestration size of different organs. The mRNA included in the LNP composition may also depend on one or more desired delivery targets. For example, mRNA can be selected for a particular indication, condition, disease, or disorder, and/or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., local or specific delivery).

The amount of mRNA in a lipid composition may depend on the size, sequence, and other characteristics of the mRNA. The amount of mRNA in the LNP can also depend on the size, composition, desired target, and other characteristics of the LNP composition. The relative amounts of mRNA and other elements (e.g., lipids) may also vary. The amount of mRNA in the LNP composition can be measured, for example, using absorption spectroscopy (e.g., uv-vis spectroscopy).

In some embodiments, the one or more mRNA, lipid, and polymer, and amounts thereof, may be selected to provide a particular N: P ratio (ratio of positively chargeable lipid or polymeric amine (n=nitrogen) groups to negatively charged nucleic acid phosphate (P) groups). The N: P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the mRNA. In general, a lower N to P ratio is preferred. The ratio N to P may depend on the particular lipid and its pKa. In certain embodiments, the mRNA and the LNP composition and/or relative amounts thereof can be selected to provide an N to P ratio of from about 1:1 to about 30:1 or from about 1:1 to about 20:1. In certain embodiments, the N to P ratio may be, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1. In certain embodiments, the N to P ratio may be from about 2:1 to about 5:1. In certain embodiments, the N to P ratio may be about 4:1. In other embodiments, the N to P ratio is from about 4:1 to about 8:1. For example, the N to P ratio may be about 4:1, about 4.5:1, about 4.6:1, about 4.7:1, about 4.8:1, about 4.9:1, about 5.0:1, about 5.1:1, about 5.2:1, about 5.3:1, about 5.4:1, about 5.5:1, about 5.6:1, about 5.7:1, about 6.0:1, about 6.5:1, or about 7.0:1.

The amount of mRNA in the nanoparticle composition can depend on the size, sequence, and other characteristics of the mRNA. The amount of mRNA in a nanoparticle composition may also depend on the size, composition, desired target, and other characteristics of the nanoparticle composition. The relative amounts of mRNA and other elements (e.g., lipids) may also vary. In some embodiments, the wt/wt ratio of the lipid component to mRNA in the nanoparticle composition can be from about 5:1 to about 50:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1. For example, the wt/wt ratio of the lipid component to mRNA may be from about 10:1 to about 40:1. The amount of mRNA in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., uv-vis spectroscopy).

Encapsulation efficiency of mRNA describes the amount of mRNA that is encapsulated or otherwise associated with a lipid composition after preparation relative to the initial amount provided. Encapsulation efficiency is desirably high (e.g., near 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of mRNA in a solution containing the lipid composition before and after decomposing the LNP composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free mRNA in a solution. For LNP compositions of the invention, the encapsulation rate of mRNA can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In certain embodiments, the encapsulation efficiency may be at least 80%.

VI formulation and delivery modes

The LNP composition of the invention can be formulated, in whole or in part, as a pharmaceutical composition. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidelines for formulating and manufacturing pharmaceutical compositions and medicaments are available, for example, in Remington's (2006) supra. Conventional excipients and adjunct ingredients may be used in any pharmaceutical composition of the invention unless any conventional excipient or adjunct ingredient may be incompatible with one or more components of the LNP composition of the invention. If the combination of excipients or adjunct ingredients with the components of the LNP composition can result in any undesirable biological or otherwise deleterious effect, it may be incompatible with the components.

In some embodiments, one or more excipients or adjunct ingredients can comprise greater than 50% of the total mass or volume of a pharmaceutical composition comprising an LNP composition of the invention. For example, the one or more excipients or adjunct ingredients can comprise 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In certain embodiments, the excipient is approved, for example, by the U.S. food and drug administration for use in humans and for veterinary use. In certain embodiments, the excipient is pharmaceutical grade. In certain embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), the European Pharmacopeia (EP), the british pharmacopeia, and/or the international pharmacopeia.

The relative amounts of the one or more lipids or LNP, one or more pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical composition will vary depending on the identity, size, and/or condition of the subject being treated and further depending on the route of administration of the composition.

The lipid composition and/or pharmaceutical composition comprising one or more LNP compositions can be administered to any subject, including human patients that can benefit from the therapeutic effect provided by the delivery of a nucleic acid, such as RNA (e.g., mRNA, tRNA, or siRNA), to one or more specific cells, tissues, organs, or systems, or groups thereof (e.g., the renal system). Although the description provided herein of LNP compositions and pharmaceutical compositions comprising LNP compositions is primarily directed to compositions suitable for administration to humans, the skilled artisan will appreciate that such compositions are generally suitable for administration to any other mammal. It is understood that the composition suitable for administration to humans is modified so as to render the composition suitable for administration to a variety of animals.

Pharmaceutical compositions according to the present disclosure may be prepared, packaged and/or sold in bulk as single unit doses and/or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of an active ingredient (e.g., a payload).

The pharmaceutical compositions of the present invention may be prepared in a variety of forms suitable for use in a variety of routes and methods of administration. For example, the pharmaceutical compositions of the present invention may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders and granules), dosage forms for topical and/or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches), suspensions, powders and other forms.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and/or elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.

Injectable formulations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing, wetting and/or suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension and/or emulsion in a non-toxic parenterally acceptable diluent and/or solvent, for example as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be employed include water, ringer's solution, u.s.p. And isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid find use in the preparation of injectables.

The injectable formulation may be sterilized, for example, by: filtered through a bacteria-retaining filter, and/or incorporated into a sterilant in the form of a sterile solid composition which may be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Other components

In addition, it is contemplated that the pharmaceutical composition may include one or more components other than those described above.

The pharmaceutical composition may also include one or more permeation enhancer molecules, carbohydrates, polymers, therapeutic agents, surface modifying agents, or other components. The permeability enhancer molecule may be a molecule as described in, for example, U.S. patent application publication No. 2005/0222064. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

The pharmaceutical composition may also contain surface modifying agents including, for example, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl octadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytics (e.g., acetylcysteine, mugwort (mugwort), bromelain, papain, dyers (cleodendrum), bromhexine, carbocistein, eplerenone, mesna, ambroxol, sibiriol, domino, ritostan, setronin, tiopronin, gelsolin, thymosin beta 4, alfa-streptozotase, netilmin, and erdostein), and DNase (e.g., rhDNase). The surface modifying agent may be disposed within and/or on the surface of the compositions described herein.

In addition to these components, the pharmaceutical compositions containing the LNP compositions of the invention may include any substance useful in pharmaceutical compositions. For example, the pharmaceutical composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives and other types. Excipients, such as waxes, butter, colorants, coating agents, flavoring agents and fragrances may also be included. Pharmaceutically acceptable excipients are well known in the art (see, e.g., remington's (2006) supra).

The dispersant may be selected from a non-limiting list consisting of: potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicate, sodium carbonate, crosslinked poly (vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (crosslinked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, carboxymethyl cellulose calcium, magnesium aluminum silicate Sodium lauryl sulfate, quaternary ammonium compounds, and/or combinations thereof.

Surfactants and/or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithins), colloidal clays (e.g., bentonite [ aluminum silicate ]]And[ magnesium aluminum silicate ]]) Long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glycerol monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers and carboxyvinyl polymers), carrageenans, cellulosic organisms (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose)Cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [>20]Polyoxyethylene sorbitan [ ]>60]Polyoxyethylene sorbitan monooleate [ ] and [>80]Sorbitan monopalmitate [ ] >40]Sorbitan monostearate [ ]>60]Sorbitan tristearate [ ]>65]Glycerol monooleate, sorbitan monooleate [ - ] and>80]) Polyoxyethylene esters (e.g. polyoxyethylene monostearate [ ]>45]Polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and +.>) Sucrose fatty acid ester, polyethylene glycol fatty acid ester (e.g.)>) Polyoxyethylene ethers (e.g. polyoxyethylene dodecyl ether [ ], polyoxyethylene dodecyl ether [ ]>30]) Poly (vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, < >>F 68、/>188. Cetrimide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or combinations thereof.

Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acid preservatives, and/or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulphite, sodium metabisulfite, and/or sodium sulfite. Examples of chelating agents include ethylenediamine tetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid and/or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexetidine, miconazole, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl parahydroxybenzoate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenols, phenolic compounds, bisphenols, chlorobutanol, hydroxybenzoates, and/or phenethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deferoxamine mesylate, trimethoprim bromide, butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), ethylenediamine, sodium Lauryl Sulfate (SLS), sodium Lauryl Ether Sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite.

Examples of buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium phosphate, sodium dihydrogen phosphate, sodium phosphate mixtures, tromethamine, sulfamate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, ringer's solution, ethanol, and/or combinations thereof.

In certain embodiments, the lipid nanoparticle compositions and formulations thereof are suitable for intravenous, intramuscular, intradermal, subcutaneous, intraarterial, intratumoral, or administration by inhalation. In certain embodiments, a dose of about 0.001mg/kg to about 10mg/kg is administered to a subject. Compositions according to the present disclosure may be formulated in dosage unit form to facilitate administration and uniformity of dosage. However, it should be understood that the total daily amount of the compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

The particular therapeutically effective, prophylactically effective, or otherwise appropriate dosage level (e.g., for imaging) for any particular patient will depend on a variety of factors including the severity and identity of the disorder (if any) being treated; one or more mRNAs employed; the specific composition employed; age, weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the particular pharmaceutical composition employed; duration of treatment; a medicament for combination or simultaneous use with the particular pharmaceutical composition employed; and similar factors well known in the medical arts.

VII method

The present disclosure provides methods of delivering a payload to a target cell or tissue (e.g., a target cell or tissue of a subject) and LNPs or pharmaceutical compositions containing the LNPs for use in such methods.

In certain embodiments, the invention provides methods of producing a polypeptide of interest (e.g., a protein of interest) in a mammalian cell and LNPs or pharmaceutical compositions containing the LNPs for use in such methods. Methods of producing a polypeptide in such cells involve contacting the cell with an LNP composition comprising an RNA of interest (e.g., mRNA encoding the polypeptide of interest (e.g., protein of interest)). After contacting the cell with the LNP composition, the mRNA can be taken up and translated in the cell to produce the polypeptide of interest.

In general, the step of contacting the mammalian cell with an LNP composition comprising mRNA encoding the polypeptide of interest can be performed in vivo, ex vivo, or in vitro. The amount of LNP composition contacted with the cells and/or the amount of mRNA therein can depend on the type of cell or tissue contacted, the mode of administration, the physicochemical characteristics (e.g., size, charge, and chemical composition) of the LNP composition and mRNA therein, and other factors. Typically, an effective amount of the LNP composition will allow for efficient production of the polypeptide in the cell. Efficiency metrics may include polypeptide translation (indicated by polypeptide expression), mRNA degradation levels, and immune response indicators.

The step of contacting an LNP composition comprising mRNA with a cell may involve or cause transfection, wherein the LNP composition may fuse with a cell membrane to allow delivery of the mRNA into the cell. After introduction into the cytoplasm of the cell, the mRNA is then translated into a protein or peptide via a protein synthesis machinery within the cytoplasm of the cell.

In certain embodiments, the LNP compositions described herein can be used to deliver a therapeutic or prophylactic agent to a subject. For example, mRNA included in an LNP composition can encode a polypeptide and produce a therapeutic or prophylactic polypeptide upon contact with and/or entry (e.g., transfection) into a cell. In certain embodiments, the mRNA included in the LNP compositions of the invention can encode polypeptides that can improve or increase the immunity of a subject.

In certain embodiments, contacting the cell with an LNP composition comprising mRNA can reduce the innate immune response of the cell to exogenous nucleic acids. The cell may be contacted with a first LNP composition comprising a first amount of a first exogenous mRNA, the first exogenous mRNA comprising a translatable region, and the level of innate immune response of the cell to the first exogenous mRNA may be determined. Subsequently, the cell may be contacted with a second composition comprising a second amount of the first exogenous mRNA, the second amount being a smaller amount of the first exogenous mRNA than the first amount. Alternatively, the second composition may include a first amount of a second exogenous mRNA different from the first exogenous mRNA. The step of contacting the cells with the first composition and the second composition may be repeated one or more times.

In addition, the efficiency of polypeptide production in the cell can optionally be determined, and the cell can be repeatedly contacted again with the first composition and/or the second composition until the target protein production efficiency is reached.

The present disclosure provides methods of delivering a nucleic acid (e.g., mRNA) to a mammalian cell or tissue (e.g., mammalian cell or tissue of a subject). Delivery of mRNA to such cells or tissues involves administering an LNP composition comprising the mRNA to a subject, for example, by injection (e.g., via intramuscular injection) or intravascular delivery to the subject. After administration, the LNP can be targeted to and/or contacted with a cell, e.g., an immune cell, such as a T cell. After contacting the cell with the LNP composition, the translatable mRNA can be translated in the cell to produce the polypeptide of interest.

In certain embodiments, LNP compositions of the invention can target specific types or classes of cells. Such targeting may be facilitated using the lipids described herein to form LNPs, which may also include targeting groups for targeting cells of interest. In certain embodiments, specific delivery can result in an increase in the amount of mRNA reaching a targeted target (e.g., a cell expressing or expressing at high levels a receptor of interest bound to the immune cell targeting group of the LNP) by more than 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold, as compared to other targets (e.g., cells not expressing or expressing only the receptor of interest at low levels).

The LNP compositions of the invention are useful in the treatment of diseases, disorders, or conditions characterized by a loss or abnormality in protein or polypeptide activity. Upon delivery of an mRNA encoding a deleted or aberrant polypeptide to a cell, translation of the mRNA may produce the polypeptide, thereby reducing or eliminating problems caused by the deletion of the polypeptide or by aberrant activity caused by the polypeptide. Because translation can occur rapidly, the methods and compositions of the invention are useful for treating acute diseases, disorders or conditions, such as sepsis, stroke, and myocardial infarction. The mRNA included in the LNP compositions of the invention can also alter the transcription rate of a given species, thereby affecting gene expression.

The diseases, disorders and/or conditions characterized by abnormal or abnormal protein or polypeptide activity may be administered in the compositions of the present invention, including, but not limited to, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. A variety of diseases, disorders and/or conditions may be characterized by a loss of protein activity (or a substantial reduction such that proper protein function does not occur). Such proteins may not be present, or they may be substantially nonfunctional. A specific example of a dysfunctional protein is a missense mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produces a dysfunctional protein variant of the CFTR protein, which leads to cystic fibrosis. The present disclosure provides methods for treating such diseases, disorders, and/or conditions in a subject by administering an LNP composition comprising mRNA encoding a polypeptide that antagonizes or otherwise overcomes abnormal protein activity present in cells of the subject and a lipid component comprising KL10, a phospholipid (optionally unsaturated), a PEG lipid, and a structural lipid.

The therapeutic and/or prophylactic compositions described herein can be administered to a subject in any reasonable amount and by any route of administration that is effective to prevent, treat, diagnose, or image a disease, disorder, and/or condition, and/or for any other purpose. The specific amount administered to a given subject may vary depending on the species, age and general condition of the subject, the purpose of administration, the particular composition, the mode of administration, and the like. Compositions according to the present disclosure may be formulated in dosage unit form to facilitate administration and uniformity of dosage. However, it should be understood that the total daily amount of the compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

LNP compositions comprising one or more mRNA can be administered by a variety of routes, such as oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal or intradermal, rectal, intravaginal, intraperitoneal, topical, transmucosal, nasal, intratumoral administration. In certain embodiments, the LNP composition can be administered intravenously, intramuscularly, intradermally, intraarterially, intratumorally, or subcutaneously. However, the present disclosure contemplates delivery of the LNP compositions of the invention by any suitable route (in view of possible advances in drug delivery science). Generally, the most appropriate route of administration will depend on a variety of factors, including the nature of the LNP composition comprising one or more mrnas (e.g., its stability in various bodily environments such as the blood stream and gastrointestinal tract), the condition of the patient (e.g., whether the patient is able to tolerate a particular route of administration), and the like.

In some embodiments of the present invention, in some embodiments, may be present in an amount sufficient to deliver from about 0.0001mg/kg to about 10mg/kg, from about 0.001mg/kg to about 10mg/kg, from about 0.005mg/kg to about 10mg/kg, from about 0.01mg/kg to about 10mg/kg, from about 0.05mg/kg to about 10mg/kg, from about 0.1mg/kg to about 10mg/kg, from about 1mg/kg to about 10mg/kg, from about 2mg/kg to about 10mg/kg, from about 5mg/kg to about 10mg/kg, from about 0.0001mg/kg to about 5mg/kg, from about 0.001mg/kg to about 5mg/kg, from about 0.005mg/kg to about 5mg/kg, from about 0.01mg/kg to about 5mg/kg, from about 0.05mg/kg to about 5mg/kg, from about 0.1mg/kg to about 5mg/kg, from about 1mg/kg to about 5mg/kg, from about 2mg/kg, from about 5mg/kg to about 2mg/kg, from about 5mg/kg from about 0.0001mg/kg to about 2.5mg/kg, from about 0.001mg/kg to about 2.5mg/kg, from about 0.005mg/kg to about 2.5mg/kg, from about 0.01mg/kg to about 2.5mg/kg, from about 0.05mg/kg to about 2.5mg/kg, from about 0.1mg/kg to about 2.5mg/kg, from about 1mg/kg to about 2.5mg/kg, from about 2mg/kg to about 2.5mg/kg, from about 0.0001mg/kg to about 1mg/kg, from about 0.001mg/kg to about 1mg/kg, from about 0.005mg/kg to about 1mg/kg, from about 0.01mg/kg to about 1mg/kg, from about 0.05mg/kg to about 1mg/kg, from about 0.1mg/kg to about 1mg/kg, from about 0.0001mg/kg to about 2.5mg/kg, from about 2.0001 mg/kg, from about 0.0001mg/kg to about 25mg/kg, from about 0.005mg/kg to about 25mg/kg, A dose level of a given dose of the composition from about 0.01mg/kg to about 0.25mg/kg, from about 0.05mg/kg to about 0.25mg/kg, or from about 0.1mg/kg to about 0.25mg/kg delivers a composition according to the present disclosure, wherein a dose of 1mg/kg provides 1mg of the composition per 1kg of subject body weight.

In particular embodiments, a dosage of about 0.001mg/kg to about 10mg/kg of the LNP composition of the invention may be administered. In other embodiments, a dosage of about 0.005mg/kg to about 2.5mg/kg of the LNP composition may be administered. In certain embodiments, a dose of about 0.1mg/kg to about 1mg/kg may be administered. In other embodiments, a dose of about 0.05mg/kg to about 0.25mg/kg may be administered. One or more doses may be administered daily in the same or different amounts to achieve the desired level of mRNA expression and/or therapeutic, diagnostic, prophylactic or imaging effect. The required dose may be delivered, for example, three times per day, twice per day, once per day, every other day, every third day, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, twelve, thirteen, fourteen or more administrations) may be used to deliver the desired dose. In some embodiments, a single dose may be administered, for example, before or after surgery or in the case of an acute disease, disorder, or condition.

LNP compositions comprising one or more mrnas may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. "in combination with … …" is not intended to imply that the agents must be administered simultaneously and/or formulated for delivery together, but such delivery methods are within the scope of this disclosure. For example, one or more LNP compositions comprising one or more different mrnas may be administered in combination. The composition may be administered simultaneously with, before or after one or more other desired therapeutic agents or medical procedures. Typically, each agent will be administered at a dosage and/or schedule determined for that agent. In some embodiments, the present disclosure encompasses delivery of the compositions of the present invention or imaging, diagnostic or prophylactic compositions thereof in combination with agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion and/or modify their distribution in the body.

It is further understood that the therapeutic, prophylactic, diagnostic or imaging agents utilized in combination may be administered together in a single composition or separately in different compositions. In general, it is expected that agents utilized in combination will be utilized at levels that do not exceed those when they are utilized alone. In some embodiments, the level of combined utilization may be lower than the level utilized alone.

The particular combination of therapies (therapeutic agents or procedures) to be employed in the combination regimen will take into account the compatibility of the desired therapeutic agent and/or procedure and the desired therapeutic effect to be achieved. It is also understood that the therapies employed may achieve a desired effect on the same disorder (e.g., the compositions useful for treating cancer may be administered concurrently with the chemotherapeutic agent), or they may achieve a different effect (e.g., control of any deleterious effects).

In some embodiments, no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, or no more than 50% of cells not intended to be targeted for the delivery are transfected by the LNP. In some embodiments, the cells not intended to be the target of the delivery are non-immune cells of the subject. In some embodiments, the cells not intended to be targeted for the delivery are cells not targeted by the method. In some embodiments, the cells not intended to be the target of the delivery are subject cells not targeted by the method.

In some embodiments, the half-life of a polypeptide encoded by a nucleic acid delivered to the immune cell by an LNP described herein or the nucleic acid delivered by the LNP and expressed in the immune cell is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold longer than the half-life of a polypeptide encoded by a nucleic acid delivered to the immune cell by a reference LNP or the nucleic acid delivered by the reference LNP and expressed in the immune cell.

In some embodiments, the composition of the LNP is different from the composition of the reference LNP in: the type of ionizable cationic lipid, the relative amount of ionizable cationic lipid, the length of the lipid fluke in the PEG lipid, the backbone or head group of the PEG lipid, the relative amount of the PEG lipid, or the type of immune cell targeting group, or any combination thereof. In some embodiments, the composition of the LNP differs from the composition of the reference LNP only in the type of ionizable cationic lipid. In some embodiments, the composition of the LNP differs from the composition of the reference LNP only in the amount of PEG lipid. In some embodiments, the reference LNP comprises the cationic lipid DLin-MC3-DMA or lipid 7, but is otherwise identical to the LNP tested. In some embodiments, the PEG lipid is a free PEG lipid.

In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the immune cells are transfected with the LNP. In some embodiments, the immune cell is an immune cell of a subject. In some embodiments, the immune cell is an immune cell targeted by the method. In some embodiments, the immune cell is a subject immune cell targeted by the method.

In some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher than the expression level of the nucleic acid delivered by a reference LNP. In some embodiments, expression levels are measured and compared using the methods described herein. In some embodiments, the expression level is measured by the ratio of cells expressing the encoded polypeptide. In some embodiments, the expression level is measured using FACS. In some embodiments, the expression level is measured by the average amount of the encoded polypeptide expressed in the cell. In some embodiments, the expression level is measured as the average fluorescence intensity. In some embodiments, the expression level is measured by the amount of the encoded polypeptide or other material secreted by the cell.

In some embodiments, the LNP provides at least one of the following benefits:

(iii) Increased transfection efficiency compared to reference LNP; and

In some embodiments, the LNP provides at least one of the following benefits:

(iv) Increased transfection efficiency compared to reference LNP; and

In some embodiments, the LNP provides at least one of the following benefits:

(iv) Increased transfection efficiency compared to reference LNP;

In some embodiments, the LNP provides at least one of the following benefits:

(iii) Increased transfection efficiency compared to reference LNP;

In some embodiments, the disorder is an immune disorder, an inflammatory disorder, or cancer. In some embodiments, the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine for treating or preventing a pathogen infection.

The LNP disclosed and claimed in the present disclosure is applicable to the above-described method.

Kit for use in medical applications

Another aspect of the invention provides a kit for treating a disorder. The kit comprises: an ionizable cationic lipid, a lipid-immune cell targeting group conjugate, a lipid nanoparticle composition (with or without an encapsulated payload (e.g., mRNA)) comprising an ionizable cationic lipid and/or a lipid-immune cell targeting group conjugate, and instructions for use in treating a medical disorder such as cancer or a microbial or viral infection.

Examples

The present invention will now be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

Example 1Preparation of ionizable cationic lipids

This example describes the synthesis of various cationic lipids.

Cationic lipid 1

Synthesis of cationic lipid 1 (shown below)

Prepared as described in scheme 1 below.

(scheme 1)

Ether intermediates 1-3 were prepared by: hydroxy-functional protected 1, 2-diol starting material (1-1) (0.151 mol,24 g) was reacted with dimethylaminopropyl chloride compound 1-2 (0.051 mol,24g,1 equiv.) and TBAI (0.0015 mol,554mg,0.01 equiv.) in the presence of NaOH (32%)/THF overnight at 80℃to afford ether intermediate compound 1-3 (20.1 g,0.1 mol). Thereafter, compounds 1-3 (2.2 g,0.01 mol) were deprotected (THF, 6m hcl,4 hours) to afford o-diol intermediate compounds 1-4 (1.6 g,0.009 mol) in quantitative yield. Di-acylated compound 1-4 (1.12 g, 0.006mol) using EDC (3.8 g,0.019mol,3.2 equiv.) using fatty acids 1-5 (5.9 mL,0.018mol,3 equiv.) in DCM (10 equiv.EQ DIPEA was used) to provide lipid compound 1 (238 mg,0.0034 mol). Lipid compound 1 was purified by preparative HPLC (CombiFlash Nextgen 300+Teledyne ISCO) and the final product was 98% pure (RP-HPLC-ELSD using Durashell-C18, 4.6X105 mm,3uM, catalog number DC 930505-0).

Purified lipid 1 free base (C44H 79NO5, molecular weight 702.12 g/mol) was purified in CDCl ₃ Is characterized by proton NMR spectroscopy (400 MHz), as shown in fig. 1; and characterized by LC-MS to confirm structure (NMR, m/z) and purity (NMR, LC), as shown in fig. 2A and 2B.

Cationic lipid 2

Synthesis of cationic lipid 2 (shown below)

Prepared as described in scheme 2 below.

(scheme 2)

Ester intermediate 2-3 was prepared by: the protected 1, 2-diol was acylated with dimethylaminobutyric acid compound 2-2 (0.03 mol,4.19g, 0.03eq.) using EDCl (0.03 mol,5.73g,1 eq.) in DCM (300 mL), DIPEA (0.12 mol,12.9g,4.0 eq.) and DMAP (0.006mol, 733mg,0.2 eq.) in solution1, 2-O-isopropylidene- D-Glycerol) Starting material (2-1) to give 5.44g (0.02 mol) of 2-3. Intermediate 2-3 (640 mg,0.0027mol,1.0 eq.) was deprotected in a mixture of 10mL TFA/water (50:50 v/v) and 10mL 6M HCl at room temperature to afford 560mg (0.0027 mol) of compound 2-4. Compounds 2-4 (0.003 mol,641mg,1 eq.) were esterified with fatty acids 1-5 (0.09 mol,2.52g,3 eq.) in 50mL of DCM using EDC (0.09 mol,1.72g,1 eq.) and DIPEA (0.001 mol,122mg,0.33 eq.) to provide cationic lipid 2 (260 mg,0.0003 mol).

Lipid compound 2 was purified by preparative HPLC. The resulting product was greater than 99% pure (reverse phase HPLC-ELSD using Durashell-C18, 4.6X10 mm,3uM, catalog number DC 930505-0).

Purified lipid 2 free base (C45H 79NO6, molecular weight 730.10 g/mol) was purified in CDCl ₃ Is characterized by proton NMR spectroscopy (400 MHz), as shown in fig. 3; and characterized by LC-MS to confirm structure (NMR, m/z) and purity (NMR, LC), as shown in fig. 4A and 4B.

Lipid 6 was synthesized using a method similar to that of lipid 2, except that diethylaminobutyric acid was used to generate tertiary amine headgroups, rather than dimethylaminobutyric acid for lipid 2.

Purified lipid 6 was characterized by proton NMR spectroscopy as shown in fig. 41; and characterized by mass spectrometry and reverse phase HPLC as shown in fig. 42A and 42B.

Cationic lipid 3

Synthesis of cationic lipid 3 (shown below)

Prepared as described in scheme 3 below.

(scheme 3)

Fatty acids 3-10 were prepared using scheme 3a below:

(scheme 3 a)

As shown in scheme 3a, fatty acids 3-10 (9Z, 12Z) -hexadec-9, 12-dienoic acid were synthesized using the Wittig reaction method. To produce compound 3-7, 9-bromononanoic acid (0.0148 mol,3.50 g) was reacted with PPh ₃ (0.0148 mol,3.87g,1 eq.) in 5mL toluene and refluxed for 48 hours to provide 7.23g of phosphonium bromide 3-7. To produce compound 3-9, compound 3-8 (0.0076 mol,0.87 g) was oxidized using dessmartin oxidant (0.0082 mol,3.48g,1.1 eq.) in 20mL DCM to provide aldehyde 0.85g of 3-9. Compound 3-9 (0.0076 mol,085 g) was reacted with compound 3-7 (0.0076 mol,3.78g,1 eq.) in 7.6mL of 40% NAHMDS in 60mL THF to provide 400mg fatty acid 3-10.

As shown in scheme 3, intermediate 3-3 is produced by: the free hydroxyl groups on the protected 1,2, 4-butanetriol starting material (3-1) (0.0342 mol,5.0g,1 eq.) were tosylated at room temperature with tosyl chloride (3-2) (0.034 mol,6.5g,1 eq.) and triethylamine (0.034 mol,4.9mL,1 eq.) in 250mL of DCM, DMAP (0.0111 mol,200mg,0.3 eq.). Nucleophilic displacement compound 3-3 (0.0032 mol,1.0g,1 eq.) was used with dimethylamine (0.03 mol,1.5g,10 eq.) in 16.6mL THF to provide 400mg tertiary amine 3-4. The compound 3-4 was deprotected with 2M HCl in MeOH to afford 410mg of compound 3-5. Compound 3-5 (90 mg) was esterified with fatty acids 3-10 (0.0016 mol,400mg,3 eq.) using EDC (0.0018 mol,306mg,3 eq.) in 5.4mL DCM, DIPEA (0.0024 mol,8.8mL,4.5 eq.) for 2 hours to provide 12mg of ionizable lipid 3. Lipid compound 3 was purified by preparative HPLC.

Lipid compound 3 was purified by preparative HPLC (CombiFlash Nextgen 300+Teledyne ISCO). The product purity was 99% as determined by reverse phase HPLC-ELSD (using Durashell-C18, 4.6X105 mm,3uM, catalog number DC 930505-0).

Cationic lipid 4

Synthesis of cationic lipid 4 (shown below)

Prepared as described in scheme 4 below.

(scheme 4)

Using the 9-bromononanoic acid heptanal starting material, fatty acid 9-1 was synthesized using a scheme similar to that used for the synthesis of fatty acid 4-6 above.

Double acylation of 1-4 (0.003 mol,600 mg) with EDC (0.09 mol,1.7g,3.2 eq.) in 7mL DCM, DIPEA (0.09 mol,2.45mL,3.2 eq.) provided 203mg of ionizable lipid 4 (mass, yield). Lipid compound 4 was purified by preparative HPLC (CombiFlash Nextgen 300+Teledyne ISCO) and yielded a 99% pure product (HPLC-ELSD using Durashell-C18,4.6x50mm,3um, catalog No. DC 930505-0).

Purified lipid 4 was characterized by proton NMR spectroscopy as shown in fig. 6; and characterized by mass spectrometry and reverse phase HPLC as shown in fig. 7A and 7B.

Cationic lipid 5

Synthesis of cationic lipid 5 (shown below)

Prepared as described in scheme 5 below.

(scheme 5)

As shown in scheme 5, intermediate 5-3 is produced by: free hydroxyl groups on the protected 1,2, 4-butanetriol starting material (5-1) (0.0342 mol,5.0g,1 eq.) were tosylated at room temperature using tosyl chloride (5-2) (0.039 mol,7.5g,1 eq.) and triethylamine (0.039 mol,5.6mL,1 eq.) in 250mL of DCM, DMAP (0.013 mol,230mg,0.3 eq.). Nucleophilic displacement compound 5-3 (0.0032 mol,1.0g,1 eq.) was used overnight with dimethylamine (0.03 mol,1.5g,10 eq.) in 16.6mL THF to provide 1.1g tertiary amine 5-4. Compound 5-4 (712 mg) was deprotected in 6M HCl in water (5 mL) to afford 551mg of compound 5-5. Compound 5-5 (2 g) was esterified with fatty acids 1-5 (35.5 mmol,8.88g,3 eq.) using edc.hcl (35.5 mol,6.7g,3 eq.) in 55mL DCM, DIPEA (47 mmol,6.7mL,4 eq.) for 2 hours to provide 1.09g of ionizable lipid 5. Lipid compound 5 was purified by preparative HPLC.

Lipid compound 5 was purified by preparative HPLC (CombiFlash Nextgen 300+Teledyne ISCO). The product purity was 99% as determined by reverse phase HPLC-ELSD (using Durashell-C18, 4.6X10 mm,3uM, catalog number DC 930505-0).

Purified lipid 5 free base (C ₄₂ H ₇₄ NO ₄ Molecular weight 657.57 g/mol) in CDCl ₃ As characterized by proton NMR spectroscopy (400 MHz), as shown in fig. 39A; and characterized by mass spectrometry to confirm structure (NMR, m/z) and purity (LC-ELSD), as shown in fig. 40A and 40B.

Lipid 7 was synthesized using a method similar to the synthesis of lipid 5 except that diethylamine was used in place of dimethylamine to incorporate tertiary amine headgroups.

Purified lipid 7 was characterized by proton NMR spectroscopy as shown in fig. 43; and characterized by mass spectrometry and reverse phase HPLC as shown in fig. 44A and 44B.

Scheme 6 below depicts the synthesis of lipid 9, lipid 10 and lipid 11:

(scheme 6)

Cationic lipid 9

Ester intermediate D-2 was prepared by: 4g (30.2 mmol) of the protected 1, 2-diol (1, 2-O-isopropylidene-D-glycerol) starting material (1) were acylated with dimethylaminopropionic acid compound D-1 (33.3 mmol,6.06g,1.71 eq), EDCI (33.2 mmol,6.28g,1.1 eq), DIPEA (121.0 mmol,21.08mL,4.0 eq) and DMAP (6 mmol,740mg,0.2 eq) in DCM (100 mL) to give 2.6g (0.02 mol) of D-2 in 37% yield. Intermediate D-2 (2.5 g,10.68mmol,1.0 eq.) was deprotected in a 1:3 (v/v) mixture of 1M aqueous HCl and THF (total volume 20 mL) at room temperature to afford 2.4g (12.6 mmol) of crude compound D-3. Crude compound D-3 (12.6 mmol,2.4g,1 eq.) was esterified with fatty acid 2 (8.9 g,2.5eq,31.8 mmol), EDCI (6.1 g,2.5eq,31.8 mmol), DIPEA (5.53 mL,2.5eq,31.8 mmol), DMAP (284 mg,0.2eq,2.6 mmol), DCM (100 mL) to provide 7g of crude ionizable lipid 9. The crude product (3 g) was purified by preparative HPLC to give 100mg of pure lipid (> 99% pure as measured by reverse phase HPLC-ELSD (using Durashell-C18, 4.6X105 mm,3uM, cat. DC 930505-0).

Purified lipid 9 free base (C44H 77NO6, molecular weight 716.10 g/mol) was characterized by proton NMR spectroscopy (400 MHz) (in CDCl 3) and mass spectrometry to confirm structure (fig. 48A and 48B), and by LC-ELSD to determine purity (fig. 48C).

Cationic lipid 10

Ester intermediate E-2 was prepared by: 1.9g (7.38 mmol) of E-2 were obtained in 25% yield from 4g (30.2 mmol) of protected 1, 2-diol (1, 2-O-isopropylidene-D-glycerol) starting material (1) acylated with dimethylaminopropionic acid compound E-1 (4.76 g,1.1eq,33 mmol), EDCI (6.38 g,1.1eq,33 mmol), DIPEA (21.08 mL,4.0eq,120 mmol) and DMAP (680 mg,0.2eq,6 mmol) in DCM (150 mL). Intermediate E-2 (1.9 g,7.38mmol,1 eq) was deprotected in a 1:3 (v/v) mixture of 1M aqueous HCl and THF (total volume 80 mL) at room temperature to afford 1.58g (7.27 mmol) of crude compound E-3. Crude compound E-3 (7.27 mmol,1.58g,1 eq.) was esterified with fatty acid 2 (18.2 mmol,5.1g,2.5 eq.), EDCI (18.2 mmol,3.48g,2.5 eq.), DIPEA (18.2 mmol,3.16mL,2.5 eq.) and DMAP (1.4 mmol,160mg,0.2 eq.) in DCM (80 mL) to provide about 7.5g of crude ionizable lipid 10. The crude product (3 g) was purified by preparative HPLC to give 105mg of pure lipid (> 99% pure as measured by reverse phase HPLC-ELSD (using durshell-C18, 4.6x50mm,3um, cat# DC 930505-0).

Purified lipid 10 free base (C46H 79NO6, molecular weight 742.14 g/mol) was characterized by proton NMR spectroscopy (400 MHz) (in CDCl 3) and mass spectrometry to confirm structure (fig. 49A and 49B), and by LC-ELSD to determine purity (fig. 49C).

Cationic lipid 11

Ester intermediate F-2 was prepared by: 4g (30.2 mmol) of the protected 1, 2-diol (1, 2-O-isopropylidene-D-glycerol) starting material (1) were acylated with dimethylaminopropionic acid compound F-1 (6.06 g,1.38eq,41.7 mmol), EDCI (6.38 g,1.1eq,33.3 mmol), DIPEA (21.08 mL,4.0eq,121.0 mmol) and DMAP (740 mg,0.2eq,6 mmol) in DCM (100 mL) to give 3.3g (12.7 mmol) of F-2 in 41.7% yield. Intermediate F-2 (3.2 g,12.3mmol,1 eq) was deprotected in a 1:3 (v/v) mixture of 1M aqueous HCl and THF (total volume 80 mL) at room temperature to afford 3.1g (14.1 mmol) of crude compound F-3. Crude compound F-3 (14.13 mmol,3.1g,1 eq.) was esterified with fatty acid 2 (35.3 mmol,9.9g,2.5 eq), EDCI (6.8 g,2.5eq,35.3 mmol), DIPEA (6.2 mL,2.5eq,35.3 mmol) and DMAP (316 mg,0.2eq,2.8 mmol) in DCM (100 mL) to provide about 9g of crude ionizable lipid 11. The crude product (3 g) was purified by preparative HPLC to give 55mg of pure lipid 11 (> 99% pure as measured by reverse phase HPLC-ELSD (using Durashell-C18, 4.6X105 mm,3uM, catalog number DC 930505-0)).

Purified lipid 11 free base (C46H 81NO6, molecular weight 744.16 g/mol) was characterized by proton NMR spectroscopy (400 MHz) (in CDCl 3) and mass spectrometry to confirm structure (fig. 50A and 50B), and by LC-ELSD to determine purity (fig. 50C).

Scheme 7 and scheme 8 below depict the synthesis of lipids 12 and 13:

(scheme 7)

(scheme 8)

Cationic lipid 12

Fmoc protected intermediate 3A was produced from (R) - (2, 2-dimethyl-1, 3-dioxolan-4-yl) methanol (1) 2.0g (1.0 eq,15.2 mmol) using Fmoc chloride (30 mmol,7.9g,2.0 eq) in pyridine (20 mL) to afford 3.8g of 3A in 71% yield (step 1, scheme 7). 3A 2.3g (1.0 eq,6.5 mmol) was selectively deprotected in 1M HCl: THF (1:3, 20 mL) and 0.5mL methanol to provide about 2g of 4A (step 2, scheme 7). 1.3g (1.0 eq,4.2 mmol) of 4A was O-acylated with linoleic acid 1-5 (9.2 mmol,2.9mL,2.2 eq) using PyBOP (9.2 mmol,4.7g,2.2 eq) and DIPEA (9.2 mmol,1.6mL,2.2 eq) in 3mL DMF. The combined products from the two batches provided a total of 1.6g (21%) of pure intermediate H-8' (step 3, scheme 7). Removal of Fmoc (step 4, scheme 7) (0 ℃ C., 4 hours) from H-8'2.05g (1.0 eq,2.44 mmol) using 1% piperidine in THF (25 mL) produced 680mg of purified intermediate H-9 in 45% yield. The key intermediate H-9 is used in subsequent steps to produce lipid 12 (via O-acylation of G-4 ') and to produce lipid 13 (via O-acylation of H-5', as described in the relevant section below).

Protected compound G-4'3- ((2- ((tert-butyldimethylsilyl) oxy) ethyl) (methyl) amino) propanoic acid was prepared via michael addition using starting materials methyl acrylate H-2' and 2- (methylamino) ethan-1-ol G-1 '. Methyl acrylate (H-2 ') 1.6ml (1 eq,17.8 mmol) was reacted with G-1' (2G, 1.5eq,26.6 mmol) and alumina (284 mg,0.5eq,8.9 mmol) at room temperature under solvent-free conditions for 3 hours to provide 2.58G (91%) of G-2' (step 7, scheme 8). The conversion of G-2'1.33G (1 eq,8.26 mmol) to tert-butyldimethylsilyl-protected intermediate G-3' (step 8, scheme 8) was carried out overnight at room temperature using tert-butyldimethylsilyl chloride TBDMSCl (1.62G, 1.3eq,10.74 mmol) and TEA (2.3 ml,2eq,16.52 mmol) in 3ml DCM, resulting in recovery of about 1.13G (50%) of purified G-3'. Subsequent selective deprotection of 1.14G (1 eq,4 mmol) of G-3 'overnight at room temperature in THF/MeOH/1M HCl (3/2/1 (v/v); total volume 6 ml) yielded 1.13G of G-4' (step 9, scheme 8). G-4' and H-9 are combined to yield intermediate G-6 (step 5A, scheme 8). H-9 400mg (1.0 eq,0.65 mmol) was acylated with G-4' (0.98 mmol,268mg,1.5 eq), DIPEA (167. Mu.L, 1.5eq,0.98 mmol), DMAP (15.9 mg,0.2eq,0.13 mmol) in 2.0mL DCM (198mg, 1.5 eq), to give 308mg (55%) of crude G-6. Deprotection of crude G-6 (308 mg) in HF. Pyridine (9.0 mmol, 641. Mu.L, 25 eq) in 6.0mL THF (step 6A, scheme 7) yields 308mg of crude lipid 12. The crude product was purified twice using preparative HPLC to isolate 213mg (79%) of purified lipid 12. (> 99% pure as measured by reverse phase HPLC-ELSD (using Durashell-C18, 4.6X10 mm,3uM, catalog number DC 930505-0)).

Purified lipid 12 free base (C45H 79NO7, molecular weight 746.13 g/mol) was characterized by proton NMR spectroscopy (400 MHz) (in CDCl 3) and mass spectrometry to confirm structure (fig. 51A and 51B), and by LC-ELSD to determine purity (fig. 51C).

Cationic lipid 13

Protected compound 3- (bis (2- ((tert-butyldimethylsilyl) oxy) ethyl) amino) propionic acid (H5 ') was prepared via michael addition using starting materials methyl acrylate H-2' and 2,2 '-azetidinediylbis (ethan-1-ol) H-1'. Methyl acrylate (H-2 ') 1.65g (1 eq,19.2 mmol) was reacted with H-1' (28.5 mmol,3.0g,1.5 eq), alumina (960 mg,0.5eq,9.6 mmol) at room temperature under solvent-free conditions for 3 hours to provide 3.53g (97%) of H-3'. H-3'830mg (1 eq,4.3 mmol) was converted overnight at room temperature to tert-butyldimethylsilyl-protected intermediate H-4' using tert-butyldimethylsilyl chloride TBDMSCl (1.56 g,2.5eq,10.9 mmol) in 8mL DCM, TEA (1.16 mL,2.0eq,10.9 mmol), resulting in recovery of about 1.51g (83%) of purified H-4'. Subsequent selective deprotection of H-4'600mg (1 eq,1.4 mmol) in THF (3 ml)/MeOH (2 ml)/1M LiOH (1 ml) at room temperature overnight yielded about 500mg of H-5'. Intermediate H-10 is produced by O-acylating H-9 using 3- (bis (2- ((tert-butyldimethylsilyl) oxy) ethyl) amino) propanoic acid (H5'). H-5' and H-9 are combined to yield intermediate H-10 (step 5, scheme 7). EDCI (0.36 mmol,74mg,1.5 eq), DIPEA (0.36 mmol, 62. Mu.L, 1.5 eq), DMAP (0.048 mmol,6mg,0.2 eq) in 1.0mL DCM was used to acylate H-9 150mg (1.0 eq,0.24 mmol) with H-5' (0.36 mmol,150mg,1.5 eq) to provide 108mg (44%) of crude H-10. Deprotection of crude H-10 108mg (1.0 eq,0.11 mmol) in HF. Pyridine (2.75 mmol, 200. Mu.L, 25 eq) in 2.0mL THF yielded 41mg (48%) of crude lipid 13. The crude products from the two batches were combined and purified twice using preparative HPLC to isolate 71mg of purified lipid 13. (> 99% pure as measured by reverse phase HPLC-ELSD (using Durashell-C18, 4.6X105 mm,3uM, catalog number DC 930505-0)).

Purified lipid 13 free base (C46H 81NO8, molecular weight 776.15 g/mol) was characterized by proton NMR spectroscopy (400 MHz) (in CDCl 3) and mass spectrometry to confirm structure (fig. 52A and 52B), and by LC-ELSD to determine purity (fig. 52C).

Example 2Preparation of LNP by vortex mixing using exemplary ionizable lipids

Exemplary LNPs were generated using cationic lipid 2 and cationic lipid 5, synthesized as in example 1, and commercially available cationic lipid 8 and cationic lipid DLin-MC3-DMA (MedChemExpress; catalog number HY-112758, N.J.).

LNP with encapsulated mRNA payload and lipid blend was produced by vortexing the aqueous mRNA solution and the ethanol lipid solution. mRNA (9:1 w/w mixture of mRNA encoding eGFP and eGFP mRNA labeled with Cy5, triLink Biotechnologies, calif.) was mixed with pH 4 acetate buffer to provide a final aqueous mRNA solution containing 133. Mu.g/mL mRNA and 21.7mM acetate buffer. The lipid components were dissolved in absolute ethanol in the relative ratios set forth in table 3 below.

TABLE 3 Table 3

Briefly, mRNA solution (375. Mu.L) was transferred to a conical bottom centrifuge tube and lipid solution (125. Mu.L) was added quickly to the tube containing mRNA solution (v/v ratio of mRNA solution to lipid solution 3:1). Immediately, the tube containing the mixture was capped and vortexed at 2,500rpm for 15s, then incubated at room temperature for no less than 5min, followed by ethanol removal and buffer exchange.

After mixing, the resulting LNP suspension was ethanol stripped and buffer exchanged by gravity flow using a Sephadex G-25 resin-filled SEC column (PD MiniTrap G-25, cytiva, mass.). Briefly, the SEC column was washed five times with 2.5mL of exchange buffer (25 mM pH 7.4HEPES buffer with 150mM NaCl) and then loaded with 425. Mu.L of LNP suspension. Once the LNP suspension is completely transferred into the resin bed, 75 μl of stacking volume of exchange buffer is applied to the column according to manufacturer's instructions to achieve the designated target loading volume of the column and maximize recovery. After the packed volume was completely transferred into the resin bed, the SEC column was transferred into a new centrifuge tube and the LNP suspension was eluted by adding 1.0mL of exchange buffer to the column. The eluate containing LNP in exchange buffer was recovered and stored at 4 ℃ until further use.

Example 3Characterization of LNP

This example describes characterization of the LNP produced in example 2.

The LNP samples produced in example 2 were characterized to determine the average hydrodynamic diameter, zeta potential and mRNA content (total sum dye accessible). Hydrodynamic diameter was determined by Dynamic Light Scattering (DLS) using Zetasizer model ZEN3600 (Malvern Pananalytical, uk). Zeta potential was measured by laser doppler electrophoresis using a Zetasizer in 5mm pH 5.5mes buffer and 5mm pH 7.4hepes buffer.

The RNA content of the nanoparticles was measured using a Thermo Fisher Quant-iT riboGreen RNA assay kit. Dye accessible RNAs (which include both unincorporated RNA and RNA near the nanoparticle surface) were measured by: nanoparticles were diluted to approximately 1 μg/mL mRNA using HEPES buffered saline, and then Quant-iT reagent was added to the mixture. The total RNA content was measured by: particles were diluted to 1 μg/mL mRNA using HEPES buffered saline, the nanoparticles were destroyed by heating to 60 ℃ for 30 minutes in HEPES buffered saline containing 0.5% triton, and then Quant-It reagent was added. RNA was quantified by measuring fluorescence at 485/535nm and concentration was determined relative to a standard curve of RNA run simultaneously. The results are set forth in Table 5.

TABLE 5

Example 4-Preparation of Fab conjugates to achieve T cell targeting

This example describes the generation of exemplary lipid-immune cell targeting group conjugates.

anti-CD 3 Fab (hSP with mouse λ and human λ, see amino acid sequence below) was conjugated to DSPE-PEG (2 k) -maleimide via covalent coupling between maleimide groups and C-terminal cysteines in Heavy Chains (HC). The anti-CD 3 Fab clone Hu291, anti-CD 8 Fab clone TRX2, anti-CD 8 Fab clone OKT8, nonfunctional mutant OKT8 (mutOKT 8), anti-CD 4Fab from the ibalizumab sequence, anti-CD 5 Fab clone He3, anti-CD 7 Fab clone TH-69, anti-CD 2 Fab clone TS2/18.1, anti-CD 2 Fab clone 9.6, anti-CD 2 Fab clone 9-1 with human kappa were also conjugated using similar methods described herein. After buffer exchange into anaerobic pH 7 phosphate buffer, the protein (3-4 mg/mL) was reduced in 2mM TCEP in anaerobic pH 7 phosphate buffer at room temperature for 1 hour. Reduced protein was separated using a 7kDa SEC column to remove TCEP, and buffer exchanged into fresh anaerobic pH 7 phosphate buffer.

By adding DSPE-PEG-maleimide (Avanti Polar Lipids, alabama, U.S.) and 30mg/mL DSPE-PEG-OCH in an anaerobic pH 5.7 citrate buffer (1 mM citrate) ₃ (vanti Polar Lipids, alabama, usa) 10mg/mL micelle suspension (using a weight ratio of 1:1 to 1:3, depending on the protein) was used to initiate the conjugation reaction. The protein solution was concentrated to 3-4mg/mL using a 10kDa regenerated cellulose membrane followed by exchange into anaerobic pH 7 phosphate buffer using a 40kDa size exclusion column buffer. Conjugation reactions were carried out at 37℃using 2-4mg/mL protein and a 3.5 molar excess of maleimide for 2 hours, followed by an additional incubation of 12-16 hours at room temperature.

The production of the resulting conjugate was monitored by HPLC and the reaction was quenched in 2mM cysteine. The resulting conjugate (DSPE-PEG (2 k) -anti hSP Fab) was isolated using pH 7.4HEPES buffered saline (25 mM HEPES, 150mM NaCl) buffer, filtered using a 100kDa Millipore regenerated cellulose membrane and stored at 4 ℃ prior to use. After quenching, the final micelle composition is composed of DSPE-PEG-Fab, DSPE-PEG-maleimide (terminated with cysteine), and DSPE-PEG-OCH ₃ Is composed of a mixture of (a) and (b). The ratio of the three components is that DSPE-PEG-Fab DSPE-PEG-maleimide (terminated with cysteine) DSPE-PEG-OCH ₃ =1:2.45:3.45-10.35 (molar).

The resulting conjugates showed comparable binding to recombinant rhesus CD3 epsilon as compared to unconjugated anti-CD 3 Fab as determined by ELISA assay.

anti-CD 3 hSP34-Fab sequence:

hSP34 HC(SEQ ID NO:1)：

hSP34-mlam LC (mouse. Lambda.) (SEQ ID NO: 2):

SP34-hlam LC (human lambda) (SEQ ID NO: 3):

anti-CD 3 Hu291-Fab sequence:

Hu291 HC(SEQ ID NO:4)：

Hu291 LC(SEQ ID NO:5)：

anti-CD 8 TRX2-Fab sequence:

TRX2 HC(SEQ ID NO:6)：

TRX2 LC(SEQ ID NO:7)：

anti-CD 8 OKT8-Fab sequence:

OKT8 HC(SEQ ID NO:8)：

OKT8 LC(SEQ ID NO:9)：

anti-CD 4 ibalizumab-Fab sequence:

ibalizumab HC (SEQ ID NO: 10):

abamectin LC (SEQ ID NO: 11):

anti-CD 5 He3-Fab sequence:

He3 HC(SEQ ID NO:12)：

He3 LC(SEQ ID NO:13)：

anti-CD 7 TH-69-Fab sequence:

TH-69HC(SEQ ID NO:14)：

TH-69LC(SEQ ID NO:15)：

anti-CD 2 TS2/18.1-Fab sequence:

TS2/18.1HC(SEQ ID NO:16)：

TS2/18.1LC(SEQ ID NO:17)：

anti-CD 2 9.6-Fab sequence:

9.6 HC(SEQ ID NO:18)：

9.6 LC(SEQ ID NO:19)：

anti-CD 2 9-1-Fab sequence:

9-1 HC(SEQ ID NO:20)：

9-1LC(SEQ ID NO:21)：

mutOKT8-Fab sequence:

mutOKT8 HC(SEQ ID NO:22)：

mutOKT8 LC(SEQ ID NO:23)：

DIVMTQSPSSLSASVGDRVTITCRTSRSISAALAWYQEKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

example 5Preparation of LNP containing T cell targeting group

This example describes the incorporation of an exemplary immune cell targeting conjugate into preformed LNP.

The LNP from example 3 and the conjugate from example 4 were combined in Eppendorf tubes as shown in table 6 and vortexed at 2,500rpm for 10 seconds. Eppendorf tubes were placed in a ThermoMixer at 300rpm for 14 hours at 37℃and then stored at 4℃until use.

TABLE 6

This example describes the incorporation of immune cell targeting conjugates into preformed LNPs.

LNP from example 2 and the conjugate prepared using the method described in example 4 (anti-CD 3 (hSP) and anti-CD 8 (TRX-2) conjugates) were combined in Eppendorf tubes as shown in table 6A and vortexed at 2,500rpm for 10 seconds. Eppendorf tubes were placed in a ThermoMixer at 300rpm for 14 hours at 37℃and then stored at 4℃until use. Alternatively, an Eppendorf tube was placed in a thermo Mixer at 300rpm for 30 minutes to 3 hours at 60℃and then mixed further for 12-24 hours at 4℃and 300rpm and then stored at 4℃until use.

TABLE 6A

Example 6Preparation of LNP by microfluidic mixing using exemplary ionizable lipids

This example describes the preparation of LNP by a microfluidic mixing method using cationic lipid 5 and cationic lipid 8.

LNP with encapsulated mRNA payload and lipid blend was produced by mixing an aqueous mRNA solution and an ethanol lipid solution using an in-line microfluidic mixing method. mRNA (9:1 w/w mixture of mRNA encoding eGFP and eGFP mRNA labeled with Cy5, triLink Biotechnologies, calif.) was mixed with pH4 acetate buffer to provide a final aqueous mRNA solution containing 133. Mu.g/mL mRNA and 21.7mM acetate buffer. The lipid components were dissolved in absolute ethanol in the relative ratios set forth in table 7 below.

TABLE 7

mRNA and lipid solutions were mixed using a NanoAssemblr Ignite microfluidic mixing device (product model NIN 0001) and an NxGen mixing cartridge (product model NIN 0002) from Precision Nanosystems inc (No. columbia, canada). Briefly, mRNA and lipid solutions were each loaded into separate polypropylene syringes. The mixing cartridge is inserted into NanoAssemblr Ignite and then the syringe is attached to the cartridge. Both solutions were then mixed with a lipid solution (325. Mu.L) using NanoAssemblr Ignite an mRNA solution (975. Mu.L) at a 3:1v/v ratio at a total flow rate of 9 mL/min. The resulting suspension was incubated at room temperature for not less than 5min, followed by ethanol removal and buffer exchange.

After mixing, the resulting LNP suspension was ethanol stripped and buffer exchanged by gravity flow using two Sephadex G-25 resin-filled SEC columns (PD MiniTrap G-25, cytiva, mass.). Briefly, SEC columns were each washed five times with 2.5mL of exchange buffer (25 mM pH 7.4hepes buffer with 150mM NaCl) and then each column was loaded with 450 μl of LNP suspension. Once the LNP suspension was completely transferred into the resin bed, a stacking volume of 50 μl of exchange buffer was applied to each column according to manufacturer's instructions to achieve the designated target loading volume of the column and maximize recovery. After the packed volume was completely transferred into the resin bed, the SEC columns were transferred into new centrifuge tubes and the LNP suspension was eluted by adding 1.0mL of exchange buffer to each column. The LNP-containing eluate in exchange buffer was recovered from each column, pooled into a single LNP batch, and stored at 4 ℃ until further use.

The resulting LNP was characterized as described in example 3. The results are summarized in table 8 below. As seen in table 8, the microfluidic method resulted in particles below 100nm that exhibited narrow polydispersity and good mRNA encapsulation (< 20% dye accessible RNA).

TABLE 8

Example 7Characterization of LNP pKa using toluidinyl-naphthalene sulfonate (TNS) fluorescent probes

This example describes the apparent pK for measurement of lipid nanoparticles _a Is based on fluorescent dyes. Apparent pK _a The nanoparticle surface charge under physiological pH conditions is determined, and pKa values typically in the endosomal pH range (6-7.4) result in LNP being neutral or slightly charged in the plasma or extracellular space (pH 7.4) and becoming strongly positive in an acidic endosomal environment. This positive surface charge drives fusion of the LNP surface with the negatively charged endosomal membrane, leading to destabilization and rupture of the endosomal compartment and escape of the LNP into the cytoplasmic compartment, a key step in mRNA and protein expression via the conjunctive cytoplasm of the cellular ribosomal machinery.

The apparent pKa of LNPs prepared using ionizable lipids 2, 5 (synthesized as described in example 1), 6 and 7 (synthesized using a method similar to lipids 2, 5, respectively, except that diethylamine was used in place of dimethylamine to incorporate tertiary amine headgroups) was determined by performing 6- (p-toluidinyl) -2-naphthalenesulfonic acid (TNS) fluorescence measurements in an aqueous buffer covering a range of pH values (pH 4-pH 10). TNS does not fluoresce when free in solution, but emits strong fluorescence when associated with positively charged lipid nanoparticles. At pH values below the pKa of the nanoparticles, positive LNP surface charge causes dye recruitment at the particle interface, producing TNS fluorescence. At pH values above the LNP pKa, no fluorescence was observed. The apparent pKa of the LNP is reported as the pH at which fluorescence reaches 50% of its maximum, as determined using a four-point logistic curve fit. Lipid 2 exhibited an apparent pKa of 7.5, and chemical modification of the tertiary amine head group in lipid 6 resulted in a shift in pKa to a lower value (lipid 6pKa about 7, fig. 8A). Similarly, lipid 5 exhibited an apparent pKa of 6.9, and chemical modification of the tertiary amine head group in lipid 7 resulted in a shift in pKa to a lower value (lipid 7pKa about 6.3, fig. 8B). As a result, both modifications were found to produce LNP that may be able to improve the ability to fuse with negatively charged endosomal membranes and result in improved cytoplasmic delivery of mRNA payloads.

Example 8In vitro transfection protocol in primary human T cells

This example describes a protocol for in vitro LNP transfection in primary human T cells. This method was used to assess the in vitro efficacy of LNP in relevant target cells (cd3+ T cells) by: cells were first transfected with LNP loaded with mRNA encoding a reporter gene (e.g., GFP mRNA), and then transfection was assessed by measuring reporter gene expression via Fluorescence Activated Cell Sorting (FACS). In addition, the association of particles with cells can be observed in the same assay by: individual nanoparticle components (e.g., mRNA) were labeled with fluorescent dyes (e.g., cy 5) and then cell/dye associations were observed by FACS.

Cd3+ T cells were isolated from frozen peripheral blood mononuclear cells using EasySep human T cell isolation kit on RoboSep automated cell isolation system from stem mel. T cells were plated into RPMI cell culture medium supplemented with glutamax, 10% fetal bovine serum, and 40ng/mL IL2 in flat bottom 96-well plates. Each well was seeded with 100 μl of cell suspension at a density of 1M T cells/mL (100K T cells/well). Cells were allowed to stand in an incubator at 37℃for two hours and then transfected by gentle addition of 10. Mu.L of 22. Mu.g/mL (as mRNA) nanoparticle suspension, resulting in a final mRNA concentration of 2. Mu.g/mL. The cells were gently mixed with a pipette and then incubated in an incubator at 37℃for 24 hours. After incubation, cells were analyzed using a ThermoFisher Attune NXT flow cytometer. Cy5 was detected using a 638nm laser with a 670/14nm filter. eGFP was detected using a 488nm laser and a 530/30nm filter. The data was analyzed using FlowJo software from BD biosciences. FACS data was first gated to exclude bimodal and dead cells, then gated for GFP and Cy5. Gating for GFP+ and Cy5+ was set such that the control samples (PBS-treated T cells) were ∈0.1% positive.

Example 9Lipid 2, lipid 6LNP properties and in vitro protein expression in primary human T cells

This example describes the transfection capacity of LNP derived from lipid 2 and lipid 6. The nanoparticle is produced first using a mixing method, followed by buffer exchange. The resulting particles were then tested in vitro in human cd3+ T cells to assess LNP association with cells and expression of reporter genes.

Lipid 2 and lipid 6LNP encapsulated a 90-10 (w/w) mixture of GFP-mRNA and cyanine-5 dye-labeled mRNA (TriLink Biotechnologies inc.) were prepared using the mixing method described in example 6, the buffer exchange method described in example 21 below. Both formulations gave particles exhibiting hydrodynamic diameters in the range below 150nm and moderate polydispersity, as well as good mRNA encapsulation and recovery (< 25% dye accessible mRNA and >80% encapsulated mRNA recovered using Triton de-formulation procedure described in example 3).

As seen in fig. 9A and 9B and table 9, moderate changes in particle size and PDI were observed after insertion of the anti-CD 3 hSP-PEG 2k-DSPE conjugate using the insertion procedure described in example 4. The resulting targeted LNP was evaluated in primary human T cells using the in vitro transfection protocol described in example 8. As seen in fig. 11, both formulations were well tolerated by T cells at doses below 0.5 μg/mL (cell viability decreased <40% relative to PBS control), with lipid 6LNP resulting in moderately higher viability at higher doses of 2 μg/mL. Dose-dependent expression of GFP protein was observed with both ionizable lipids (2 and 6), as demonstrated by a high percentage of gfp+ cells and strong GFP MFI values. As illustrated by Cy5+ and Cy5 MFI values, both formulations were associated equally with cells, indicating that the conjugate insertion process was independent of the chemical composition of the ionizable lipids. Both ionizable lipids (2 and 6) gave acceptable mRNA encapsulation levels (< 30% dye accessible RNA and >60% total mRNA recovery).

TABLE 9 lipid 2 and lipid 6LNP mRNA levels

These findings demonstrate that lipid nanoparticles prepared using alternative ionizable lipids 2 and 6 can effectively encapsulate mRNA and transfect T cells in vitro.

Example 10Lipid 5, lipid 7LNP profile and in vitro protein expression in primary human T cells

This example describes the transfection capacity of LNP derived from lipid 5 and lipid 7. The nanoparticle is produced first using a mixing method, followed by buffer exchange. The resulting particles were then tested in vitro in human cd3+ T cells to assess LNP association with cells and expression of reporter genes.

Lipid 5 and lipid 7LNP encapsulated a 90-10 (w/w) mixture of GFP-mRNA and cyanine-5 dye-labeled mRNA (TriLink Biotechnologies inc.) were prepared using the mixing method described in example 6, the buffer exchange method described in example 21 below. Both formulations gave particles exhibiting hydrodynamic diameters in the range below 150nm and moderate polydispersity, as well as good mRNA encapsulation and recovery (< 25% dye accessible mRNA and >80% encapsulated mRNA recovered using Triton de-formulation procedure described in example 3). As seen in fig. 10A and 10B and table 10, lipid 5LNP exhibited a large hydrodynamic diameter change (relative to lipid 7 LNP) following insertion of the anti-CD 3 hSP-PEG 2k-DSPE conjugate using the insertion procedure described in example 4. The resulting targeted LNP was evaluated in primary human T cells using the in vitro transfection protocol described in example 8. As seen in fig. 12A-12E, both formulations were well tolerated by T cells at doses equal to and below 0.5 μg/mL (minimal decrease in cell viability was observed relative to PBS control). As illustrated by Cy5+ and Cy5 MFI values, both formulations were associated equally with cells, indicating that the conjugate insertion process was independent of the chemical composition of the ionizable lipids.

Dose-dependent expression of GFP protein was observed with both ionizable lipids (5 and 7), as illustrated by similar% gfp+ and GFP MFI values (fig. 12A and 12B). Both formulations gave similar levels of cell association as illustrated by similar% Cy5+ and Cy5 MFI values (fig. 12C and 12D). However, the lipid 7LNP formulation (apparent pKa about 6.4) exhibited significantly reduced GFP protein expression levels relative to the lipid 5LNP formulation (apparent pKa about 7), indicating that the cytoplasm in the case of lipid 7LNP is accessible relatively poorly. Both ionizable lipids (5 and 7) gave acceptable mRNA encapsulation levels (< 30% dye accessible RNA and >60% total mRNA recovery).

TABLE 10 lipid 5 and lipid 7LNP mRNA levels

Example 11In vitro protein expression in primary human T cells, lipid 5, lipid 8 and DLn-MC3-DMA LNP Properties

This example compares the GFP protein expression generated by LNPs derived from lipid 5 and lipid 8 with LNPs prepared using DLn-MC 3-DMA. The nanoparticle is produced first using a mixing method, followed by buffer exchange. The resulting particles were then tested in vitro in human cd3+ T cells to assess LNP association with cells and expression of reporter genes.

Lipid 5, lipid 8 and DLn-MC3-DMA LNP encapsulated a 90-10 (w/w) mixture of GFP-mRNA and cyanine-5 dye labeled mRNA (TriLink Biotechnologies inc.) were prepared using the vortexing and buffer exchange methods described in example 4. All three formulations gave particles exhibiting hydrodynamic diameters and moderate polydispersity in the range below 150nm (fig. 37A and 37B). In addition, all three formulations prepared using lipids 5, 8 and DLn-MC3-DMA using the vortexing method showed acceptable mRNA encapsulation (< 30% dye accessible mRNA) and moderate mRNA recovery (> 60% of the encapsulated mRNA was recovered using the Triton de-formulation procedure described in example 3). As seen in fig. 37, insertion of the anti-CD 3 hSP34-PEG2k-DSPE conjugate using the insertion procedure described in example 4 resulted in only a slight increase in hydrodynamic diameter and PDI. The resulting targeted LNP was evaluated in primary human T cells using the in vitro transfection protocol described in example 8. As seen in fig. 38, all three formulations were well tolerated by T cells at doses below 0.5 μg/mL (cell viability decreased <40% relative to PBS control), with lipid 8LNP exhibiting slightly decreased viability at higher doses of 2 μg/mL (fig. 38E). Dose-dependent expression of GFP protein was observed with ionizable lipids 2 and 8, as demonstrated by high percentages of gfp+ cells and strong GFP MFI values (fig. 38A, 38B). However, DLn-MC3-DMA (also shown in FIGS. 38A, 38B) LNP failed to express GFP protein. Comparison of Cy5+ and Cy5 MFI values in lipid 2 or lipid 8 formulations with corresponding levels in DLn-MC3-DMA LNP transfection (FIGS. 38C and 38D) indicated that all three formulations were equally associated with T cells, indicating that the efficiency of the antibody insertion process was independent of the chemical composition of the ionizable lipids. The poor performance of DLn-MC3-DMA LNP may be due to poor cytoplasmic availability of mRNA in primary human T cells as a result of this formulation.

TABLE 11 lipid 5, lipid 8 and DLn-MC3-DMA LNP mRNA content

Example 12 in vitro protein expression-CD 3 and CD8 targeting Cy5/GFP LNP of various densities

This example describes targeting human CD 8T cells with anti-CD 3 or anti-CD 8 Fab inserted into Cy5/GFP mRNA LNP after various Fab densities and their effects on particle binding, transfection, viability, CD69 up-regulation and ifnγ secretion.

LNP was prepared using the mixing method described in example 6, the buffer exchange method described in example 21. hSP34 and TRX2 Fab-lipid conjugates produced by the method described in example 4, as well as non-T cell specific anti-HER2 lipid-conjugates (Nellis DF, ekstrom DL, kirpotin DB, zhu J, andersson R, broadt TL, ouelette TF, perkins SC, roach JM, drummond DC, hong K, marks JD, park JW and Giarina SL (2005) Preclinical manufacture of an anti-HER2 scFv-PEG-DSPE, liponame-insertion con-ugate.1.gram-scale production and purification.Biotechnol Prog 21:205-220) were post-inserted at various densities (SP 34 0.6-17g/mol; TRX2 3-9g/mol; anti-HER2 17 g/mol) into LNP containing mRNA marked with lipids 8, 5 and GFP (GFP: mass ratio Cy 1: 9): the conjugate and LNP were added together and the solution was heated in a thermocycler at 60 ℃ for 60min without mixing and cooled to 4 ℃ for 3-5min. The pellet was then diluted to 25 μg/mL mRNA with Hepes buffered saline pH 7.4, and human CD 8T cells were then transfected for about 24 hours using a method similar to example 8 (with a final concentration of about 2.5 μg/mL mRNA) (or 10 μl Hepes buffered saline pH 7.4 buffer was added as mock transfection). After transfection, LNP binding efficiency (Cy 5) and transfection efficiency (GFP) were assessed by flow cytometry. The human ifnγ concentration of the supernatant was measured using a commercially available ELISA kit under conditions recommended by the manufacturer (R & D Systems Duoset).

For LNP with SP34 and TRX2 Fab inserted at a broad range of Fab densities, high transfection (fig. 13A) and binding (fig. 13B) were observed, which mediate transfection; whereas non-specific HER 2-targeted LNPs exhibit low binding and transfection. Some loss of cell viability was observed using hSP CD 3-targeted LNP (fig. 14A), whereas TRX2 CD 8-targeted LNP had similar viability to non-specific HER 2-targeted LNP and untransfected (added mock buffer) T cells. In addition, hSP (with mouse or human λ) CD 3-targeted LNP mediated high ifnγ secretion relative to TRX2 CD 8-targeted, HER 2-targeted LNP and mock T cell transfection conditions (fig. 14B).

This study showed that CD 8T cells can be efficiently transfected with CD3 and/or CD8 targeted LNP using a broad range of Fab densities in all cases. In addition, the use of anti-CD 8 Fab can mediate efficient LNP transfection while avoiding high CD69 up-regulation and ifnγ secretion.

Example 13 in vitro protein expression-CD 3, CD8 and CD3/CD8 targeting TTR-023LNP at various densities

This example describes anti-CD 3 or anti-CD 8 Fab targeting human CD 3T cells inserted into anti-CD 20 CAR (TTR-023) mRNA LNP at various Fab densities and their effect on transfection, viability, CD69 up-regulation and ifnγ secretion.

LNP was prepared using the mixing method described in example 6, the buffer exchange method described in example 21 below. hSP34 and TRX2 Fab-lipid conjugates and non-T cell specific anti-HER2 lipid-conjugates (Nellis DF, ekstrom DL, kirpothin DB, zhu J, andersson R, broadt TL, ouelette TF, perkins SC, roach JM, drummond DC, hong K, marks JD, park JW and Giarina SL (2005) Preclinical manufacture of an anti-HER2 scFv-PEG-DSPE, liposome-insertion conjugate.1.Gram-scale production and purification. Biotechnol Prog 21:205-220) were inserted at various densities (SP 34.25-17 g/mol; TRX 2.25-9 g/mol; SP34+TRX2.25-9 g/mol; anti-HER2 17 g/mol) into the target mRNA containing lipid-8 and anti-LNRT 20 CAP) using methods similar to example 12. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. For FACS analysis, in addition to staining for CD69 (Biolegend, 310930) and CD4 (Biolegend, 344648), T cells were stained with M1 antibody (Sigma, F3040) that binds to the N-terminal FLAG tag variant sequence on TTR-023CAR (sequences provided below) to distinguish CD8 cells from CD4 cells.

For hSP alone or co-targeted with TRX2, high transfection efficiencies were observed between 2-17g/mol Fab (fig. 15A and 15B), and for TRX2, transfection above background was detected at 6-9g/mol Fab. Consistent with the transfection results, CD69 was upregulated in a target-specific manner, with Fab targeting of CD3 by hSP inducing CD69 expression on both CD8 and CD4 cells, whereas targeting of CD8 by TRX2 mediated CD69 expression on only CD8 cells (fig. 16A and 16B). CD 8-targeted LNP with TRX2 Fab induced low levels of ifnγ secretion relative to CD3 and CD3/CD 8-targeted LNP (fig. 17), despite observable upregulation of CD69 on CD 8T cells.

This study showed that both CD4 and CD 8T cells could be efficiently transfected with CD 3-targeted and CD3/CD 8-targeted LNPs, and CD8 cells could be specifically transfected with CD 8-targeted LNPs (avoiding CD4 transfection) using a broad range of Fab densities in all cases. In addition, the use of anti-CD 8 Fab can mediate efficient CAR mRNA transfection while avoiding high CD69 up-regulation of ifnγ secretion.

TTR-023 anti-CD 20 (Leu-16) CAR sequence (including leader sequence) (SEQ ID NO: 24):

METDTLLLWVLLLWVPGSTGDYKAKEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

the corresponding nucleic acid sequence (SEQ ID NO: 25):

atggagaccgacaccctgttgctttgggtactgttactttgggtgcccggatctaccggtgattacaaggccaaggaggtgcagctgcagcagagcggagccgagctggtgaagccaggcgcttccgtgaagatgtcttgtaaggcctccggctacacattcaccagctacaatatgcactgggtaaagcagactccggggcagggcctggagtggataggtgccatctaccctggcaacggcgacaccagctacaaccagaagtttaaggggaaggctactctaacagcggacaagtcgtcctctaccgcctacatgcaactcagctccctgacgagcgaggactccgcggactactactgtgcccgctccaactactacggctctagctattggttcttcgacgtgtggggcgctggaacgaccgtgaccgtgtcttccggtggaggttccgggggcggaagcggcggtggcggcagttcggacatcgtgctgacccagagccctgccatcctgtccgcttccccgggggagaaagttacgatgacctgccgagcgagctccagtgtcaactacatggattggtaccagaagaagcccggcagcagtcccaagccgtggatttacgctactagcaacctggcgtccggtgtcccggctcgcttctcaggttctggctcgggtactagttattcattaaccatttctcgcgtggaggctgaggacgctgccacctactactgccaacagtggtctttcaaccctcccactttcggaggcggcaccaagctcgagatcaagggcgggggtggctccgcagcagccattgaggtgatgtatcctcctccctatttggacaacgagaagtcaaatggcaccatcatccacgttaagggcaagcacctgtgcccatctcccctgttcccaggcccctctaagcccttctgggtcctggtggtggtcggcggcgtcctggcatgttactctctgctggtgaccgtcgcgttcatcatcttttgggtccggtccaagcgcagccgcctgctccactccgactacatgaatatgactcctcgtaggcccggtccaacccgcaagcactaccagccgtacgcgccgcccagagactttgctgcttaccgatccagagtgaaattttctaggtcggccgaacctcccgcatatcagcagggccagaaccagctgtacaacgaactcaacttgggacggcgcgaggaatacgatgtgctggataaacgccgtggccgcgatcccgagatgggcgggaagccacgtcgcaaaaaccctcaggagggcctttacaacgagttgcagaaggacaaaatggcggaggcctactccgagatcggaatgaagggggagcgccggcgcggcaaagggcatgacggcctctaccagggcctgtccacagccacgaaagacacctatgacgccctgcatatgcaggccctgcccccgcgctgataatga

example 14 in vitro protein expression-CD 3 and CD8 targeting with other clones

LNP was prepared using the mixing method described in example 6, the buffer exchange method described in example 21. Using a method similar to example 12, hSP, hu291, TRX2, OKT8 Fab-lipid conjugates and non-T cell specific anti-HER2 lipid-conjugates (Nellis DF, ekstrom DL, kirpotin DB, zhu J, andersson R, broadt TL, ouellette TF, perkins SC, roach JM, drummond DC, hong K, marks JD, park JW and Giarina SL (2005) Preclinical manufacture of an anti-HER2 scFv-PEG-DSPE, liponame-insertion conjugate.1.Gram-scale production and purification. Biotechnol Prog 21:205-220) were post-inserted into LNP containing lipid 8 and Cy5/GFP mRNA at various densities (Table 11). Transfection with human CD 8T cells was performed at about 2.5 μg/mL mRNA for about 24 hours.

SP34 mediated higher% transfection (fig. 18A) and significantly higher GFP expression levels (fig. 18B) compared to Hu291 (quantified by Mean Fluorescence Intensity (MFI)), although both clones showed high levels of% Cy5+ T cells and bound to the same target CD3 (fig. 19A and 19B). Similarly, TRX2 mediated higher transfection (measured by both% gfp+ and MFI metrics) than OKT8 for CD8 targeting (fig. 18A and 18B), although both clones mediated high levels of particle binding (measured by% Cy5 ") (fig. 19A). In addition, combining OKT8 and TRX2 increased the amount of particle binding (fig. 19B), without providing enhancement of transfection (fig. 18A and 18B).

This data suggests that the epitope of Fab binding to target proteins may be important in determining their ability to mediate efficient particle uptake, transfection and translation, and that efficient binding to the target does not guarantee efficient transfection.

Table 11: post-target insertion LNP Fab density

Fab cloning	Fab Density 1 (g/mol)	Fab Density 2 (g/mol)
			SP34-mlam	12	17
Hu291	6	12
			OKT8	3	6
TRX2	3	6
			OKT8/TRX2	3	6
F5	17	-

Example 15 in vitro protein expression-CD 3, CD8, CD4 and CD8/CD4 targeting

This example shows the anti-CD 3, anti-CD 8, anti-CD 4 or anti-CD 8 and anti-CD 4 Fab targeting human CD 3T cells with insertion into Cy5/GFP mRNA LNP at various Fab densities and their effects on particle binding, transfection, viability, CD69 up-regulation and ifnγ secretion.

LNP was prepared using the mixing method described in example 6, the buffer exchange method described in example 21. Using a method similar to example 12, hSP, TRX2 and ibalizumab Fab-lipid conjugates and non-T cell specific anti-HER 2 lipid-conjugates were post-inserted into LNP containing lipid 8 and Cy5/GFP mRNA at various densities (designated in fig. 20A and 20B). Human CD 3T cells were transfected at approximately 2.5 μg/mL mRNA for approximately 24 hours and stained for CD69 (Biolegend, 310930) and CD4 (Biolegend, 344648) to distinguish CD8 cells from CD4 cells by FACS analysis.

Consistent with previous results, hSP and TRX2 mediated specific LNP binding and transfection to CD3 and CD8 cells, respectively (fig. 20A and 20B and fig. 21A, 21B). CD 4-targeted Fab based on the VH and VL sequences of ibalizumab mediate high binding and transfection of CD 4T cells while exhibiting minimal off-target binding and transfection of CD 8T cells. When TRX2 and ibalizumab were post-Fab inserted into the same LNP, high levels of binding and transfection were observed in both CD4 and CD8 cells using a broad range of Fab densities. While hSP driven high levels of CD69 upregulation (fig. 22A and 22B), TRX2 alone, ibalizumab-Fab alone, and the combination of TRX2 and ibalizumab-Fab mediated CD69 levels were much lower. In addition, SP34 driven higher levels of ifnγ compared to TRX2, ibalizumab-Fab, or combinations thereof (fig. 23).

Studies have shown that both CD4 and CD 8T cells can be efficiently transfected with CD 3-targeted and CD8/CD 4-targeted LNPs, CD8 cells can be specifically transfected with CD 8-targeted LNPs (avoiding CD4 transfection), and CD4 cells can be specifically transfected with CD 4-targeted LNPs (avoiding CD8 transfection) using a broad range of Fab densities in all cases. In addition, the use of anti-CD 8 Fab, anti-CD 4 Fab, or Fab that is both anti-CD 8 and anti-CD 4 can mediate efficient transfection while avoiding high CD69 upregulation and ifnγ secretion.

EXAMPLE 16 in vitro protocol for Whole blood transfection

This example describes a method for transfecting immune cells in whole blood using Fab-targeted mRNA LNP.

Venous blood from healthy volunteers was anticoagulated in heparin tubes (BD Biosciences # 367526) and inoculated in 96-well round bottom plates at 50 μl. Transfection of whole blood was performed simply by: nanoparticles containing 5 μg/mL mRNA were added to the cells and co-cultured at 37 ℃ until analysis. To assess transfection efficiency, cells were analyzed by flow cytometry 24 hours after transfection. LNP (with and without post-inserted target) was used at 2.5 μg/mL RDM 073.23. Cells obtained from human blood were analyzed by flow cytometry. Before analysis of whole blood transfection efficiency, erythrocytes were lysed with a VersaLyse lysis solution (Beckman coulter#a 09777) at room temperature for 10 min. Primary antibodies used in flow cytometry analysis of whole blood include the following: CD4-FITC (1:200) (BD biosciences # 555346), CD19-BUV395 (1:400) (BD biosciences # 563551), CD56-BUV737 (1:400) (BD biosciences # 741842). The viability of all samples was assessed using a fixable viability dye eFluor780 (eBiosciences # 65-0865-14). For flow analysis, 1x10 ⁵ Individual cells were Fc blocked on ice (BD Biosciences # 564219) for 5 min, then dead cells were labeled with the fixable reactive dye eFluor780, and surface stained with specific antibodies on ice for 30 min.

Each fluorescent dye was compensated in a multicolor flow panel using positive and negative compensation beads. Fluorescence Minus One (FMO) samples and unstained controls were included to determine background fluorescence levels and to gate negative and positive cell populations.

All samples were taken on BD LSRFortessa X-20 (BD Biosciences) running FACSDIVA software (Becton Dickinson). All data collected were analyzed using FlowJo 10.7.1 software and GraphPad Prism version 9.0.

Example 17 in vitro cell-specific protein expression in human Whole blood (mCherry)

This example describes the specific targeting of human T cells in whole blood with various Fab densities followed by insertion into mCherry mRNA LNP for anti-CD 3, anti-CD 8, anti-CD 2, anti-CD 5, anti-CD 7 Fab or combinations thereof and their effect on transfection and CD69 up-regulation secretion.

LNP was prepared using the vortex mixing method described in example 2 using the component ratios described in table 12 below. The conjugate from the method described in example 4 was inserted after particle formation. The particle characteristics were characterized using the method described in example 3 and are described in table 13 below.

Table 12

* Depending on the particular conjugate. TRX2 was used at 0.6 nmol/100. Mu.g mRNA.

TABLE 13

Batch of	Size (nm)	PDI	EE(％)	Zeta (mV) at pH 7.4
					73.23	123.6	0.217	93.2	N.D.

Using the methods described in example 16, the transfection efficiency of classical ionospheric formulations with the insertion (density depicted in table 14) of Fab clone hSP (anti-CD 3), TRX2 (anti-CD 8), he3 (anti-CD 5), anti-CD 2 (TS 2, 9.6 or 9-1), anti-CD 7 (TH-69) or mutOKT8, and combinations of these, respectively, was measured directly in human whole blood. LNP with lipid 8 was transfected with 2.5. Mu.g/mL mCherry mRNA in WB for 24 hours.

TABLE 14

Conjugate 1	Conjugate 2	Density (g/mol)	Target 1	Target 2
					hSP34	-	9,00	CD3	-
TRX2	-	9,00	CD8	-
					TRX2	hSP34	9,00	CD8	CD3
He3	-	3,00	CD5	-
					He3	TRX2	3,00	CD5	CD8
TS2/18.1	-	1,50	CD2	-
					9.6	-	1,50	CD2	-
TS2/18.1	TRX2	1,50	CD2	CD8
					9.6	TRX2	1,50	CD2	CD8
TH-69	-	3,00	CD7	-
					TH-69	TRX2	3,00	CD7	CD8
9-1	TS1/18.1	1,50	CD2	CD2
					9-1	9.6	1,50	CD2	CD2
He3	TH-69	3,00	CD5	CD7
					mutOKT8	-	9,00	NT	-
DSPE-PEG	-	9,00*	NT	-
					TH-69	hSP34	3,00	CD7	CD3
TS2/18.1	He3	1,50	CD2	CD5

* DSPE-PEG addition amount matching the amount of Fab addition of about 48kD from 9g/mol

Depending on the target and clone, all targeted fabs achieved observable transfection against the background with varying degrees of efficiency. The most efficient transfection of both CD8 and CD4 cells was observed using the combinations of hSP, he3, and hSP34/He3, he3/TH-69, hSP34/TRX2, he3/TRX2, hSP/TH-69, TH-69/TRX2, 9.6/9-1, TS2/9-1 (FIGS. 24A and 24B). TRX2 effectively transfected CD 8T cells, whereas no transfection was observed in CD 4T cells (fig. 24A and 24B). For the combination with TRX2/He3, 9.6/9-1, TS2/9-1 and He3/TH-69, additive effects were observed in terms of transfection efficiency (FIGS. 24A and 24B), indicating that synergistic effects can be mediated by targeting two different targets or two different epitopes on the same target. In addition to their combination, transfection was also observed in NK cells with CD3, CD8, CD2 and CD7 targeting (fig. 25B). Overall, no off-target transfection was observed in B cells (fig. 25A) and granulocytes (fig. 26A). High CD69 upregulation was observed in CD4 and CD8 cells alone in the case of CD3 targeting or a combination of CD3 targeting with other targets like CD8 or CD7 (fig. 26B and 26C). In addition, non-targeted LNP with similar DPSE-PEG inserted after Fab targeting formulations and LNP with mutOKT8 Fab inserted after did not show transfection of any immune cell type, indicating that specific transfection was mediated by Fab targeting (fig. 24A and 24B).

This study shows that in whole blood, CD4 and CD 8T cells can be efficiently transfected with CD 3-, CD 5-, CD 7-or CD 2-targeted LNPs and targeted combinations thereof, CD8 cells can be transfected with CD 8-targeted LNP specificity (avoiding CD4 transfection), and transfection can be biased towards CD8 cells using a combination of CD8 targeting and CD 5-, CD 7-or CD 2-targeting, as compared to CD4 cells.

The combined use of Fab clones targeting different targets or Fab clones binding the same target but known to target different epitopes (e.g., anti-CD 2 clones 9.6 and 9-1) can lead to a synergistic increase in transfection efficiency. NK cells were transfected with CD8, CD7 or CD2 targeted Fab or combinations thereof, consistent with known surface expression of these markers on human NK cells or NK cell subsets. Although LNPs with anti-CD 3, anti-CD 8, anti-CD 5, anti-CD 7, or anti-CD 2 Fab, or combinations thereof, can mediate efficient transfection of T cells and NK cells (for some fabs), minimal transfection was observed in B cells or granulocytes, indicating the high specificity uptake and transfection achieved by Fab or non-targeted LNP targeting a given non-targeted Fab (mutOKT 8) does not transfect T cells or NK cells. In addition, the use of anti-CD 8, anti-CD 5, anti-CD 7 or anti-CD 2 Fab or combinations thereof may mediate efficient transfection without driving high CD69 expression.

EXAMPLE 18 in vivo reprogramming of immune cells with mCherry-expressing LNP

This example describes the time course of reprogramming immune cells in humanized mice treated with mCherry-expressing LNP.

Mouse strains and humanization

NCG mice (NOD-Prkdc) ^em26Cd52 Il2rg ^em26Cd22 NjuCrl) mouse model was purchased from Charles River Laboratories. By tail vein injectionMale mice of 4 weeks of age were transplanted with PBMC of 1000 ten thousand eligible donors (completed by Charles river) in sterile PBS and the mice were transported to the Tidal facility (facility). The body weight of the individuals was monitored twice a week and blood samples were taken at appropriate intervals to assess the engraftment of human immune cells.

Evaluation of human T cell implantation in immunodeficient mice

50ul of blood was collected from each mouse by tail vein blood sampling. Erythrocytes were lysed using Versalyse RBC lysis solution (Beckman Coulter A09777) as per the protocol indicated by the manufacturer. Cells were stained with hCD45& hCD3 to confirm engraftment of human T cells. Mice had anywhere from 30% -60% hucd45+ after PBMC injection for 30 days. The LNP expressing mCherry of these humanized mice was evaluated for reprogramming of immune cells.

Reprogramming of immune cells

At time zero, 9 mice were injected intravenously with 3mg/kg using cationic lipid 8 and the mixing method described in example 6, the buffer exchange method described in example 21 and LNP was expressed with hSP 34-lipid targeted mCherry (lot 201109APG-NF 70-409) using the method described in example 5, or 6 mice were injected with the appropriate buffer. At each time point 24, 48 and 96h, 3 mice treated with LNP or 2 mice treated with buffer were sacrificed. Peripheral blood and tissue collection was performed as follows to determine mCherry expression in different organs and immune cells.

Tissue and blood sample collection

At the time points indicated above, mice were anesthetized with CO2 prior to sample collection. For blood collection, the chest cavity is opened to expose the heart. Up to 300 μl of blood was withdrawn from the left ventricle and dispensed into a K3EDTA mini-collection tube (Greiner Bio-One). The remaining blood is then withdrawn from the heart as much as possible using a new syringe. All immune organs were isolated together with liver: spleen, bone marrow, thymus and all lymph nodes (tongue, axilla, submaxilla and mesentery). Immune cells were isolated from spleen, thymus and lymph nodes via smear and minced by syringe, and the cell suspension was filtered through a 70 μm cell strainer and washed with PBS. A piece of liver tissue was gently ground with a tissue homogenizer, and the homogenized tissue was incubated with a digestion solution (10 ml HBSS supplemented with 0.05% type IV collagenase (Sigma C5138-5G), 0.02% BSA (Sigma A2153-100G), 0.001% DNASE I (grade II, sigma 10104159001) and 1mM calcium chloride (Sigma C7902-500G) for 30min at 37 ℃. After 30min, digestion was stopped with 10ml ice-cold HBSS solution.

Immunophenotyping

Immune cells from blood and all organs above were treated with Versalyse RBC lysis buffer according to the manufacturer's instructions. Immune cells were stained with live/dead fixable dyes and surface markers using standard flow assay protocols as shown in the following set. The positive population was determined using Attune (Thermo flow cytometer).

TABLE 15 group 1

Antigens	Fluorophores	Cloning	Company (Corp)	Catalog number
					Alive read dyes	Zombie Aqua	NA	BioLegend	423102
Antihuman CD45	FITC	2D1	BioLegend	368508
					Antihuman CD3	PerCP/cyanine 5.5	UCTH1	BioLegend	300430
Antihuman CD8	APC-Fire750	SK1	BioLegend	344746
					Antihuman CD4	BV711	SK3	BioLegend	344648
Anti-human CD69	BV421	FN50	BioLegend	310930
					Antihuman CD137	BV421	4B4-1	BioLegend	309820
Antihuman 279PD-1	APC	NAT05	BioLegend	367406
					Antihuman CD366	APC	F38-2E2	BioLegend	345012
mCherry	mCherry	NA	NA	NA

TABLE 16 group 2

Antigens	Fluorophores	Cloning	Company (Corp)	Catalog number
					Alive read dyes	Zombie Aqua	NA	BioLegend	423102
Anti-human	FITC	2D1	BioLegend	368508
					Anti-human/mouse CD11b	BV785	M1/70	BioLegend	101243
Anti-mouse F4/80	APC	BM8	BioLegend	123116
					Antihuman CD19	BV711	SJ25C1	BioLegend	563036
mCherry	mCherry	NA		NA

huNCG-PBMC mice treated with mCherry expressing CD 3-targeted LNP at 3mg/kg showed mCherry in T cells of blood, liver and spleen. Cd8+ T cells (fig. 27A, 27C and 27E) showed the highest mCherry expression, with up to about 30% of cd8+ T cells in blood, liver and spleen. Cd4+ T cells (fig. 27B, 27D and 27F) exhibited mCherry expression in blood, liver and spleen up to about 15%. No reprogramming was found in the other organs analyzed. The expression of mCherry is limited to cd3+ cells. Minimal or no mCherry expression was observed in liver myeloid cells, macrophages or kupfry cells (fig. 28).

Overall, CD 3-targeted mCherry LNPs specifically reprogram T cells with minimal or no expression in myeloid populations.

Example 19-in vivo reprogramming of immune cells with mCherry or CD20 CAR expressing LNPs with CD3 and or CD8 targeting antibodies

This example describes in vivo reprogramming with LNP expressing mCherry or CD20 CAR and compares CD3 and CD8 targeting or a combination of both.

Mouse strains and humanization

NSG female mice were purchased from Jackson lab. At 8 weeks of age, mice were injected intravenously with 2000 ten thousand PBMCs (internally isolated leukopak, donor 555046,Precision for Medicine).

Evaluation of human T cell implantation in immunodeficient mice

50ul of blood was collected from each mouse by tail vein blood sampling. Erythrocytes were lysed using Versalyse RBC lysis solution (Beckman Coulter A09777) as per the protocol indicated by the manufacturer. Cells were stained with hCD45& hCD3 to confirm engraftment of human T cells. Mice had anywhere from 60% -80% hucd45+ 30 days after PBMC injection. Reprogramming of immune cells of these humanized mice was evaluated as follows.

Reprogramming immune cells with mCherry and CD20 CAR with CD3 and/or CD8 targeting antibodies

At time zero, mice (n=5) were injected intravenously with i) buffer or LNP expressing the following at a dose of 3 mg/kg; ii) TTR-023mRNA targeted with 17g/mol hSP 34; iii) TTR-023mRNA targeted with 9g/mol hSP; iv) TTR-023mRNA, targeting with 9g/mol TRX 2; v) TTR-023mRNA targeted with 9g/mol TRX2+9g/mol hSP; vi) mCherry mRNA (n=3 mice), targeted with 17g/mol hSP 34. After 24h, 50ml of blood were collected and processed as mentioned in example 18. At 96h, mice were injected with a second dose of LNP (as above) or buffer at 3 mg/kg. Following the second dose, peripheral blood and organs were collected and treated as described in example 18 at the time point of 40 h.

LNP was prepared using the mixing method described in cationic lipid 8 and example 6, the buffer exchange method described in example 21 below, and targeted with hSP-lipid or TRX 2-lipid using the method described in example 5. The following table summarizes the formulations and lot numbers used.

Table 17.

Immunophenotyping

Similar immunophenotyping assays were performed as described in example 18 in the groups listed below. CD20 CAR expression was assessed by detecting the M1 tag expressed by the CD20 CAR with a first M1 antibody, followed by a second antibody.

TABLE 18 group 1

TABLE 19 group 2

Antigens	Fluorophores	Cloning	Company (Corp)	Catalog number
					Alive read dyes	Zombie Aqua	NA	BioLegend	423102
Anti-human	FITC	2D1	BioLegend	368508
					Anti-human/mouse CD11b	BV786	M1/70	BioLegend	101243
Anti-mouse F4/80	BV421	BM8	BioLegend	123132
					Antihuman CD19	BV711	SJ25C1	BioLegend	563036
mCherry	mCherry	NA	NA	NA
					M1 tag	NA	NA	Sigma	M1-F3040
Secondary antibodies to M1	APC	NA	Southern biotech	1090-11S

After 24h of the first dose of 3mg/kg, anti-CD 3 targeting was used,>60% of the T cells were reprogrammed with mCherry mRNA (FIG. 29A). Both CD3 and CD8 targeting showed>20% of T cells reprogrammed with CD20CAR mRNA, while the combination of the two showed about 30% of T cells reprogrammed with CD20CAR mRNA (fig. 29B). After 3mg/kg of the second dose of anti-CD 20CAR expressed LNP 40h, targeting with CD3,>30% of T cells were reprogrammed with anti-CD 20 CAR; spleen>Blood>Liver>Bone marrow>Thymus (fig. 30A-30E). No significant increase in T cell reprogramming was observed with increasing anti-CD 3 targeting density (fig. 30A-30E). After 3mg/kg of the second dose of anti-CD 20CAR expressed LNP 40h, targeting with CD8 showed the greatest reprogramming in the spleen compared to other tissues>50%) (fig. 30A-30E). As expected, CD8 targeting selectively targets cd8+ T cellsThe cells are reprogrammed. The combination of CD3 and CD8 targeting showed the most robust reprogramming in a variety of tissues; blood = spleen >Bone marrow>Thymus gland>Liver (fig. 30A-30E). After 40h of the second dose, anti-CD 3 targeting was used,>60% of T cells are coveredmCherry mRNAReprogramming; spleen>Liver>Bone marrow>Blood (fig. 31A-31E). At this time, no reprogramming was observed in thymus or lymph nodes in the case of mCherry expressing LNP.

Overall, there was a difference in the distribution of CD3 or CD8 targeted reprogrammed cells, which also appeared to be dependent on mRNA cargo. Without wishing to be bound by theory, this is also likely due to the different kinetics of redistribution of T cells that have been reprogrammed in the periphery and other organs. CD8 targeting shows specificity for reprogramming of CD 8T cells in blood and all organs tested. No reprogramming was observed in myeloid cells in blood or organs.

Example 20: LNP pharmacokinetic studies in mice

This example describes the pharmacokinetics of LNP in female BALC/c mice.

LNP was prepared using the vortex mixing method described in example 2 using the component ratios described in table 20 below. The particle characteristics were characterized using the method described in example 3 and are described in table 21 below.

Table 20.

Table 21

Batch of	Size (nm)	PDI	EE(％)
				83.1	157.4	0.165	79.6*

* Encapsulation efficiency may be affected by DiI-C18 (3) -DS fluorescence interfering with mRNA quantification

8 female BALB/c mice were purchased from Janvier Labs (Le Genest-Saint-Isle, france) and were acclimatized for one week. Food was served ad libitum.

Mice were intravenously injected 3mg/kg single dose of LNP formulated with 1,1' -octacosyl-3, 3' -tetramethylindocarbocyanine-5, 5' -disulfonic acid (DiI-C18 (3) -DS) and mCherry mRNA via the tail vein. Blood samples were obtained from the facial veins and collected at times ranging from 30 minutes to 24 hours (n=2 for each time point). The samples were centrifuged at 10,000g x 10 min and the serum was stored at 4 ℃ until the analysis time. The pharmacokinetics of LNP for the formulations described in table 20 were determined in Balb/c mice after blood collection at the time points outlined in fig. 32 and are shown in fig. 33, where LNP was only cleared slowly from circulation over 24 h.

Fluorescence quantification was performed using a fluorescence microplate reader (Spark multimode microplate reader, tecan). The reading is from the top, where the excitation/emission wavelength is 555/570nm. The nanoparticles in the cycle were quantified by interpolation using a standard curve.

The theoretical initial LNP concentration in serum was calculated to be 37.5 μg/mL using an average mouse weight of 25g and an approximate blood volume of 2mL, with about 80% of the injected dose detected in plasma after 30min, about 40% of the injected dose detected in plasma after 1 hour, and about 10% of the injected dose detected in plasma after 8 hours (fig. 33).

The present study shows that current formulations can maintain circulating mRNA levels above 0.5 μg/mL for more than 24 hours when administered at a mRNA dose of 3 mg/kg.

Example 21-preparation of LNP by microfluidic in-line mixing and tangential flow filtration Using exemplary ionizable lipids

This example describes the preparation of LNP using scalable unit operations (i.e., online microfluidic mixing followed by Tangential Flow Filtration (TFF) for ethanol removal and buffer exchange).

Using the mixing method of example 6, multiple LNP batches were pooled together, totaling 60mL, with a concentration of RNA of 300. Mu.g/mL. Ethanol removal and buffer exchange were then performed using Tangential Flow Filtration (TFF).

After mixing, the resulting LNP suspension was ethanol removed and buffer exchanged using a hollow fiber TFF module (Repligen, U.S. Pat. No. 5, N D02,02-E100-05-N). Briefly, TFF modules were rinsed with DI water and drained prior to use. LNP was then added to the reservoir and an exchange buffer (25 mM ph7.4hepes buffer with 150mM NaCl) was used as diafiltration buffer. The TFF module was started and Diafiltration (DV) was then started by: the peristaltic pump was ramped up to the target flow rate and the retentate valve was adjusted until the target transmembrane pressure (TMP) was reached. Once diafiltration has been initiated, the target operating parameters of the system are a flow rate of 212mL/min and a TMP of 3.5 psi. The TMP was kept constant throughout the diafiltration by adjusting the retentate valve. Permeate flow rate was monitored and it did not decrease significantly over time. Six diafiltrations were performed, with the sample set aside at the end of each diafiltration, in order to follow the buffer exchange process later. The final ethanol content was <0.1%, as measured by refractive index measurement of DV samples, and pH measurement confirmed buffer exchange into exchange buffer. After six diafiltrations were completed, the pump was stopped and the resulting LNP suspension was subsequently concentrated.

Concentration of LNP suspension was performed using the same TFF module used during the buffer exchange procedure. After ramping the pump, the TMP and flow rate from the buffer exchange process are maintained and the suspension is allowed to concentrate by stopping the addition of diafiltration buffer. The resulting LNP suspension was collected and filtered with a 0.2 μm syringe filter. The suspension was sampled for analytical purposes and then stored at 4 ℃ until further use.

Using the LNP characterization method in example 3, LNP batches were characterized to determine average hydrodynamic diameter and mRNA content (total and dye accessible); is set forth in table 22 below. As seen in table 22, the microfluidic mixing method with ethanol removal and buffer exchange by TFF resulted in particles below 100nm that exhibited narrow polydispersity and good mRNA encapsulation (< 20% dye accessible RNA).

Table 22

Example 22: role of PEG in Whole blood transfection of mRNA LNP

This example describes anti-CD 3 Fab specific targeting of human T cells in mCherry mRNA LNP with or without DiR labeling and incorporating varying levels of PEG during particle formation with post-insertion in whole blood to determine the effect of PEG on LNP binding (DiR signal) and transfection efficiency (mCherry).

The SP34-Fab lipid conjugates from the method described in example 4 were 3 PEG-lipid variants (DSPE-PEG 2k-Fab, DSPE-PEG2 k-maleimide (quenched), DSPE-PEG2 k-OCH) ₃ ) Thus exploring the effect of additional PEG (DMG-PEG 200). LNP was prepared using the vortex mixing method described in example 2 using the component ratios described in table 23 below, and the conjugate was inserted after particle formation. The particle characteristics were characterized using the method described in example 3 and are described in table 24 below.

Table 23.

Table 24.

Batch of	Size (nm)	PDI	EE(％)
				RDM085.8	103.2	0.113	96.3
RDM0138	113.8	0.096	87.4
				RDM073.19	119.4	0.129	83.4
RDM149	170.5	0.083	92.3

Venous blood from healthy volunteers was anticoagulated in hirudin tubes (sarsed # 04.1959.001) and inoculated at 50 μl into 96-well round bottom plates. Transfection of whole blood was performed by: LNP containing 2.5. Mu.g/mL mRNA was added to the cells and co-cultured at 37℃until analysis. To assess the binding of DiR-labeled LNP, cells were analyzed 2 hours after LNP addition and the transfection efficiency of cells was analyzed by flow cytometry after 24 hours of incubation.

For non-targeted LNPs, DSPE-PEG2k was only post-inserted to match the SP 34-targeted LNP, labeled DSPE-PEG in fig. 34A-36B. For non-targeted LNPs, no detectable binding (fig. 34A-35B) or transfection (fig. 36A and 36B) was observed, indicating that SP34 mediated highly specific transfection via CD3 targeting. For particles inserted after the SP34-Fab lipid conjugate, LNP lacking DMG-PEG200 during particle formation exhibited higher binding (as measured by DiR signal) to CD4 (fig. 34A and 34B) and CD8 (fig. 35A and 34B) T cells, but transfection efficiency was reduced (fig. 36A and 36B) compared to LNP containing DMG-PEG200 during particle formation.

This study shows that the introduction of the 4 th PEG-lipid variant into the particles can have a significant effect on particle uptake and transfection efficiency in addition to the 3 PEG-lipid variants added as part of the post-insertion process, in which case a difference of approximately 2-3 fold is observed in transfection efficiency.

Example 23-lipid 8 and lipid 5LNP Properties, in vitro cell viability and protein expression in Primary human T cells

This example describes the relative in vitro toxicity of CD 3-targeted LNPs derived from lipid 8 and lipid 5 in primary human T cell transfection of GFP-mRNA. The nanoparticle is produced first using a mixing method, followed by buffer exchange. The resulting particles were then tested in vitro in human cd3+ T cells to assess T cell viability, LNP association with cells, and reporter gene expression at three LNP doses.

Lipid 8 and lipid 5LNP were prepared using the mixing method described in example 6, the buffer exchange method described in example 21, encapsulating a 90-10 (w/w) mixture of GFP-mRNA and cyanine-5 dye-labeled mRNA (TriLink Biotechnologies inc.). Lipid 5LNP was produced using lipid 5 stock solutions (lipid 5 (O) and lipid 5 (N), respectively) that had been stored frozen at-20 ℃ for 2 weeks or 1 day. Both formulations gave particles exhibiting hydrodynamic diameters and moderate polydispersity in the range below 100nm and good mRNA encapsulation and recovery (table 25 <25% dye accessible mRNA and ≡80% encapsulated mRNA recovered using Triton de-formulation procedure described in example 3). As seen in fig. 45A and 45B, lipid 5LNP exhibited a large hydrodynamic diameter change (relative to lipid 8 LNP) following insertion of the anti-CD 3 hSP34-PEG2k-DSPE conjugate using the insertion procedure described in example 4. The resulting targeted LNP was evaluated in primary human T cells using the in vitro transfection protocol described in example 8. As seen in fig. 46A-46E, the PBS control group exhibited about 50% T cell viability, while lipid 8LNP at 0.125, 0.5, and 2ug mRNA/mL doses per well exhibited dose-dependent toxicity to T cells, with T cell viability decreasing from about 45% survival at 0.125ug mRNA/mL per well to about 25% survival at 2ug mRNA/mL per well. In contrast, lipid 5LNP (both samples "O" and "N") was consistently better tolerated by T cells, with 40% -45% T cell viability observed at all three dose levels. The lower toxicity observed with lipid 5LNP may be due to faster degradation and clearance of lipid 5 from T cells driven by hydrolysis and/or enzymatic degradation of labile ester bonds in the lipid 5 molecule.

Dose-dependent expression of GFP protein was observed with both ionizable lipids (5 and 8), however, as demonstrated by both%gfp+ and GFP MFI values (fig. 46A and 46B), lipid 5LNP resulted in higher overall protein expression at all three mRNA dose levels, indicating that lipid 5LNP improved cytoplasmic availability of mRNA payload.

TABLE 25 lipid 8 and lipid 5LNP mRNA levels

Overall, these data indicate that CD 3-targeted LNPs formed with lipid 5 show both lower cytotoxicity and higher transfection activity in human T cells compared to LNPs prepared with lipid 8.

EXAMPLE 24 Standard procedure for in vivo reprogramming of immune cells with GFP-expressing DiI LNP

The following standard procedure for in vivo reprogramming of immune cells with GFP-expressing DiI LNP was used in the experiment in example 29.

Mouse strains and humanization

NSG (NOD.Cg-Prkdcsccid Il2rgtm1 Wjl/SzJ) mouse model was purchased from Jax Laboratories. PBMCs of 1000 ten thousand eligible donors in sterile PBS were implanted by tail vein injection into 6-8 week old male mice. The body weight of the individuals was monitored twice a week and blood samples were taken at appropriate intervals to assess the engraftment of human immune cells.

Evaluation of human T cell implantation in immunodeficient mice

50ul of blood was collected from each mouse by tail vein blood sampling. Erythrocytes were lysed using Versalyse RBC lysis solution (Beckman Coulter A09777) as per the protocol indicated by the manufacturer. Cells were stained with hCD45& hCD3 to confirm engraftment of human T cells. Mice had anywhere from 30% -60% hucd45+ 15 days after PBMC injection. The reprogramming of immune cells by LNP expressing DiI dye and GFP was evaluated in these humanized mice.

Reprogramming of immune cells

At time zero, mice (n=4 per group) were injected intravenously with GFP expressing DiI LNP at 0.3mg/kg or with the appropriate buffer at 0.1 mg/kg. At each time point 24 or 48h, mice treated with LNP or buffer were sacrificed, depending on the example. Peripheral blood and tissue collection was performed as follows to determine the expression of DiI and GFP in different organs and immune cells.

Tissue and blood sample collection。

At the time points indicated above, CO was used prior to sample collection ₂ Mice were anesthetized. For blood collection, the chest cavity is opened to expose the heart. Up to 300 μl of blood is withdrawn from the left ventricle and dispensed to K ₃ EDTA micro-collection tubes (Greiner Bio-One). The remaining blood is then withdrawn from the heart as much as possible using a new syringe. All are separated together with liver and lung Immune organ: spleen, bone marrow. Immune cells were isolated from the spleen via smear and minced by syringe, and the cell suspension was filtered through a 70 μm cell strainer and washed with PBS. Bone marrow was flushed with a needle to collect all immune cells. A piece of liver and lung tissue was gently ground with a tissue homogenizer, and homogenized cells were isolated using Miltenyi liver dissociation kit (Miltenyi Biotec, catalog No. 130-105-807) and lung dissociation kit (Miltenyi Biotec, catalog No. 130-0950927), and following the instructions for manufacture.

Immunophenotyping

Immune cells from blood and all organs above were treated with Versalyse RBC lysis buffer according to the manufacturer's instructions. Immune cells were stained with live/dead fixable dyes and surface markers using standard flow assay protocols as shown in the following set. Positive populations were determined using BD symphony flow cytometer.

Group of

EXAMPLE 25 alternative ethanol removal and buffer exchange methods

In addition to the ethanol removal and buffer exchange methods described in example 6, alternative processes may be used to produce the LNP of the present disclosure. Specifically, after mixing, the resulting LNP suspension was subjected to ethanol removal and buffer exchange using a discontinuous diafiltration process. A centrifugal ultrafiltration device (Amicon Ultra-15, millipore Sigma, mass.) with a 100,000kDa MWCO regenerated cellulose membrane was sterilized with 70% ethanol solution and then washed twice with exchange buffer (25 mM pH 7.4HEPES buffer with 150mM NaCl). LNP suspension (1.5 mL) was then loaded into the device and centrifuged at 500rcf until the volume was reduced by half (0.75 mL). The suspension was then diluted with exchange buffer (0.75 mL) to bring the suspension back to its original volume. This two-fold concentration and two-fold dilution process was repeated five more times for a total of six discrete diafiltration steps. The retentate containing LNP in exchange buffer was recovered from the centrifugal ultrafiltration device and stored at 4 ℃ until further use.

Example 26 lipid 8 and lipid 5LNP Properties, and in vitro cell viability and protein expression in Primary human T cells

This example compares the properties of LNP prepared using lipid 5 and lipid 8 and GFP protein expression in primary human T cells. Two LNP formulations were prepared using the microfluidic mixing method as described in example 6 and using the discontinuous diafiltration method for ethanol removal as described in example 25 (table 26). LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa) and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa) using the lipid ratios shown in table 26 below. The LNP insertion targeting conjugate is then used using the specified conditions to provide the final targeted LNP formulation. The LNP was characterized as described in example 3. The characteristics of the LNP are shown in table 27.

Table 26 lnp formulation composition and antibody conjugate insertion conditions

TABLE 27 LNP size, charge (dynamic light scattering) and mRNA encapsulation (Ribogreen assay)

* mRNA levels determined using the Triton De-formulation procedure described in example 3

Both lipid 5 and lipid 8 formulations gave particles exhibiting hydrodynamic diameters in the range below 100nm (table 27 and fig. 53A) with a narrow polydispersity (< 0.1) before antibody conjugate insertion and moderately higher polydispersity (< 0.3) after antibody conjugate insertion. In addition, low levels of dye accessible mRNA (< 15%) and high RNA encapsulation efficiency (> 80% mRNA recovered in the final formulation relative to the total RNA used in LNP batch preparation) were observed in both formulations. These improvements in LNP size distribution are due to the use of discontinuous diafiltration to remove ethanol (described in example 25) as opposed to ethanol removal by buffer exchange using size exclusion columns (described in example 6). As seen in fig. 53B and 53C, both lipid 5 and lipid 8LNP exhibited positive zeta potentials at pH 5.5 and near neutral or slightly negative charges at pH 7.4 before and after antibody insertion (fig. 53D), indicating that the LNP ionization state was changed as expected.

The resulting targeted LNP was evaluated in primary human T cells using the in vitro transfection protocol described in example 8. Dose-dependent expression of GFP protein was observed with both ionizable lipids (5 and 8), as illustrated by similar% gfp+ and GFP MFI values (fig. 54A and 54B). However, a two-fold higher mean fluorescence intensity (GFP MFI) was observed with lipid 5LNP (at both 0.5ug/mL and 1.0ug/mL dose/well), indicating that cytoplasmic release of mRNA payload was more efficient (and hence GFP protein expression was higher) with lipid 5 formulation relative to lipid 8 formulation. As illustrated by the dii+ and DiI MFI values, both formulations were associated equally with cells, indicating that the conjugate insertion process was independent of the chemical composition of the ionizable lipids (fig. 54C and 54D). As seen in fig. 54E, both formulations were well tolerated by T cells at doses equal to and below 1.0 μg/mL (minimal decrease in cell viability was observed relative to PBS control).

Example 27-lipid 5, lipid 8 and DLn-MC3-DMA LNP Properties and in vitro GFP protein expression in Primary human T cells

This example compares the properties of LNP prepared using lipid 5, lipid 8 with the properties of DLn-MC3-DMA LNP and in vitro GFP protein expression in primary human T cells. All LNP formulations were prepared using a microfluidic mixing method (described in example 6) and using a discontinuous diafiltration method for ethanol removal (described in example 25) (table 28). LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa) and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa) using the lipid ratios shown in table 28 below. The LNP insertion targeting conjugate is then used using the specified conditions to provide the final targeted LNP formulation. The LNP was characterized as described in example 3.

TABLE 28 LNP formulation composition and antibody insertion conditions

TABLE 29 LNP size, charge (dynamic light scattering) and mRNA encapsulation (Ribogreen assay)

DLn-MC3-DMA, lipid 5 and lipid 8 were formulated using 1.5 mol% DPG-PEG. As seen in table 29 and fig. 55A-55B, all LNPs exhibited hydrodynamic Diameters (DLS) below 100nm prior to antibody insertion and about 100nm or less after antibody conjugate insertion. Lipid 5LNP polydispersity was still narrow (< 0.15) after antibody conjugate insertion, while DLn-MC3-DMA and lipid 8 showed slightly more varied polydispersity (about 0.2) after insertion. In addition, low levels of dye accessible mRNA (< 15%) and high RNA encapsulation efficiency (> 80% mRNA in the parental LNP sample) were observed in all formulations (table 29 and fig. 55D). As shown in fig. 55C, all three formulations exhibited positive zeta potential at pH 5.5 and near neutral or slightly negative charge at pH 7.4 prior to antibody insertion, indicating a change in LNP ionization state as expected using the in vitro transfection protocol described in example 8 to evaluate the resulting targeted LNP in primary human T cells.

As seen in fig. 56E, all formulations were well tolerated by T cells at doses below 0.125 μg/mL (similar to PBS control). However, both ester-based lipids DLn-MC3-DMA and lipid 5 were better tolerated at higher doses, with lipid 5 being least toxic at the 1ug/mL dose. As illustrated by dii+ and DiI MFI values (fig. 56C and 56D), all formulations showed similar levels of cell association at most of the dose levels tested, indicating that the conjugate insertion process was independent of the chemical composition of the ionizable lipids. In all cases, dose-dependent expression of GFP protein was observed (fig. 56A and 56B). However, at all doses tested, lipid 5 was superior to both lipid 8 and DLn-MC3-DMA, and at doses/well of 0.5ug/mL and 1.0ug/mL, the average fluorescence intensity (GFP MFI) exhibited was > 2-fold higher relative to lipid 8 and > 5-fold higher relative to DLn-MC3-DMA, indicating that cytoplasmic release of mRNA payload was more efficient (and hence GFP protein expression was higher) with lipid 5 formulation relative to both lipid 8 and DLn-MC3-DMA formulations.

Example 28 stability of lipid 5LNP formulation after freeze thawing stress

This example illustrates the stability of lipid 5LNP formulations after one freeze-thaw cycle. Lipid 5LNP formulation compositions shown in table 30 were prepared using a microfluidic mixing method (described in example 6) and using a discontinuous diafiltration method for ethanol removal (described in example 25). LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa) and labeled with 0.06mol% of dic 18 (5) -DS (Invitrogen, ma). The LNP insertion targeting conjugate is then used using the specified conditions to provide the final targeted LNP formulation. The LNP size (by DLS) and mRNA content were characterized as described in example 3.

After preparation of the targeted LNP formulation, the formulation was split into two parts. One portion was exchanged into 40mM pH 7.4HEPES buffer and the other portion was exchanged into 30mM pH 7.4Tris buffer. Buffer exchange was performed using a discontinuous diafiltration process, in which LNP formulation samples were transferred to a centrifugal ultrafiltration device (Amicon Ultra-4, millipore sigma, ma) with 100,000kda MWCO regenerated cellulose membrane, then diluted 10-fold with exchange buffer, and retracted to the original volume by centrifugation at 500 rcf. This dilution and concentration step is repeated once more. The exchanged LNP samples were then split into individual aliquots, which were mixed with concentrated sodium chloride and sucrose solutions to provide the final freeze-thaw sample formulations. Samples of each freeze-thaw formulation were then stored at 4 ℃, frozen at-80 ℃, or snap frozen in liquid nitrogen. The samples were later thawed at room temperature and tested for size (by DLS) and T cell transfection in vitro.

TABLE 30 LNP formulation composition and antibody insertion conditions

TABLE 31 size and polydispersity (DLS) of lipid 5LNP prior to intercalation

TABLE 32 LNP formulation compositions and storage Condition/freezing method lists for lipid 5LNP

* LN2: liquid nitrogen

As shown in fig. 57A and 57B, the freezing method used (quick freezing in liquid nitrogen and storage in-80 ℃ refrigerator) did not affect the size distribution of LNP after freeze thawing, with both hydrodynamic radius and polydispersity trends being similar between the two methods. However, the cryoprotectant and buffer composition significantly affected the dimensional characteristics of LNP after freeze thawing. Cold storage in HEPES buffer without addition of salt or with 25 and 50mM NaCl and 9.6wt.% or 18wt.% sucrose resulted in a significant increase in LNP polydispersity. In contrast, storage in TRIS buffer and 9.6wt.% or 18wt.% sucrose is effective to preserve the size and polydispersity of the LNP relative to LNP stored at 4 ℃ without being subjected to freeze-thaw stress. All formulations were evaluated in primary human T cells using the in vitro transfection protocol described in example 8.

As seen in fig. 58A and 58B, all LNPs stored in HEPES buffer lost the ability to transfect T cells after freeze-thawing cycles, while the formulations stored in TRIS buffer retained the ability to transfect T cells after freeze-thawing activity. As seen in fig. 58C and 58D, the cell association of the HEPES buffer composition (as measured by% dii+ cells and DiI MFI values) was slightly reduced after the freeze-thaw cycle, while the cell association of the TRIS buffer formulation was maintained. The lower variation in cell association and LNP dimensional characteristics following freeze-thawing stress in HEPES buffer formulations may be responsible for the observed loss of activity. Notably, storage in TRIS buffer maintains both LNP characteristics and their ability to transfect T cells after freezing storage and freeze-thaw stress.

Example 29-PEG-lipid fluke and Effect of% PEG on in vivo reprogramming

The aim of this study was to identify the best PEG-lipids and mol% for in vivo reprogramming of immune cells. We have hypothesized that medium anchor length is optimal for T cell/NK cell or other immune cell engagement in the blood, as short chain anchors like PEG-DMPE or PEG-DMG (both C14) are lost very fast, whereas PEG-lipids with longer acyl chains like PEG-DSPE and PEG-DSG (both C18) are too stable and lead to reduced transfection efficiency.

Part A. Anti-CD 3Lipid 8LNP

In this study we used LNPs targeting CD3 (hsp 34 Fab' -PEG-DSPE conjugate) and incorporating lipid 8 to test LNPs prepared with 1.5 or 2.5mol% DMG-PEG (C14), DPG-PEG (C16), DPPE-PEG (16) or DSG-PEG (C18) based on total lipid.

LNP formulations in the following table were prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa) and labeled with 0.06mol% of dic 18 (5) -DS (Invitrogen, ma). The LNP is then inserted into the targeting conjugate using the specified conditions to provide a targeted LNP formulation. The final targeted LNP formulation was prepared by: the LNP suspension was mixed with a concentrated sucrose solution to provide the final LNP formulation with 5.3wt% sucrose. The LNP was characterized as described in example 3.

TABLE 33 formulation forms

TABLE 34 analysis results of formulations

The results of in vivo reprogramming of immune cells with CD3 targeted DiI/GFP LNP at doses of 0.3mg/kg after 2.5%24 or 48h with DMG, DPG or DSG-PEG or after 1.5% or 2.5%24h with DPPE or DSPE are shown in fig. 59A to 59T.

All formulations with anti-CD 3 hsp34 clone targeting lipid 8LNP showed peak GFP expression at 24h, with maximum expression in blood > lung > spleen > bone marrow > liver. Either the diacylglycerol or the phosphoethanolamine backbone, DPG-PEG or DPPE-PEG (i.e., C16 anchor length) showed maximum reprogramming with 1.5% PEG-lipid compared to PEG-lipids with other acyl chain lengths (C14 or C18). GFP MFI showed a similar trend as% GFP positive T cells. The percent DiI positive or DiI MFI also showed a trend similar to that of GFP positive T cells, indicating that for the CD3 targeting lipid 8lnp, the binding efficiency of lnp correlates with its reprogramming capacity.

Part B anti-CD 3 or anti-CD 8Lipid 5LNP

In this study we used lipid 5-doped, CD 3-targeting (hsp 34 Fab '-PEG-DSPE conjugate) LNP and CD 8-targeting (TRX 2 Fab' -PEG-DSPE conjugate or)V2-PEG-DSPE Nb conjugate) to test LNP prepared with 1.5mol% DMG-PEG (C14) or DPG-PEG (C16) of total lipid.

LNP formulations in the following table were prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding unmodified eGFP (TriLink Biotechnologies, california, U.S. cat.; catalog number L-7601) and labeled with 0.06mol% DiIC18 (5) -DS (Invitrogen, massachusetts, U.S.). The LNP is then inserted into the targeting conjugate using the specified conditions to provide a targeted LNP formulation. The final targeted LNP formulation was prepared by: the LNP suspension was mixed with a concentrated sucrose solution to provide the final LNP formulation with 9.6wt% sucrose. The LNP was characterized as described in example 3.

Table 35 lnp formulation

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TABLE 36 analysis results of formulations

The results of in vivo reprogramming of immune cells with 0.3mg/kg of CD3 or CD8 targeted DiI/GFP LNP of lipid 5 24h after DMG-PEG or DPG-PEG (1.5% or 2.5%) are shown in figures 60A to 60T.

Lipid 5CD 3-targeted LNP showed reprogramming of both CD4 and CD 8T cells, while CD 8-targeted antibody TRX2 or CD8 nanobody had specificity for reprogramming CD 8T cells as expected. Similarly, CD 3-targeted LNP binds to both CD4 and CD 8T cells, while CD 8-targeted LNP with antibodies or nanobodies binds only to CD 8T cells. In both the case of DMG or DPG (i.e., C14 or C16 lipid anchor length) and 1.5% PEG, CD3 targeting lipid 5LNP showed similar GFP expression, whereas in blood, at 24h, in the case of DMG-PEG, CD8 antibody or nanobody targeting LNP showed maximum GFP expression, which is 2-fold that of DPG-PEG. In other tissues (e.g., lung, spleen and bone marrow), CD 8-targeted LNP with antibodies or nanobodies showed similar GFP expression in both DMG-PEG and DPG-PEG-1.5%. GFP MFI shows a trend similar to that of GFP expression. % DiI positive T cells were observed only in blood, but not in other compartments, and DiI MFI was also maximal in blood compartments.

anti-CD 8, anti-CD 11a and anti-CD 4 lipid 5LNP

In this study we used LNP spiked with lipid 5 targeted to CD8 (nanobody-PEG-DSPE conjugate), CD11a (hmfm 24 Fab-PEG-DSPE conjugate) and CD4 (nanobody-PEG-DSPE conjugate or ibalizumab-PEG-DSPE conjugate) to test LNP prepared with 1.5mol% dpp-PEG (C16) of total lipid.

LNP formulations in the following table were prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa) and labeled with 0.06mol% of dic 18 (5) -DS (Invitrogen, ma). The LNP is then inserted into the targeting conjugate using the specified conditions to provide a targeted LNP formulation. The LNP was characterized as described in example 3.

Table 37 lnp formulation

TABLE 38 analysis results of formulations

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The results of in vivo reprogramming of immune cells with the above LNP of 0.3mg/kg of lipid 5 after 24h with DMG-PEG or DPG-PEG (1.5 mol%) are shown in FIGS. 61A-61T.

Lipid 5LNP and CD8 nanobody targeting LNP showed GFP expression specifically only in CD 8T cells but not CD 4T cells. Similarly, targeting CD4 with nanobodies or antibodies showed GFP expression specifically only in CD 4T cells, while CD11a targeting showed both GFP expressing CD4 and CD 8T cells. In the case of DMG-PEG-1.5% compared to DPG-PEG, CD8 nanobody LNP showed maximum GFP expression; whereas in both cases of DMG-PEG and DPG-PEG (1.5 mol%), both CD11a Fab and CD4 nanobodies and Fab antibodies showed similar GFP expression. GFP MFI shows a trend similar to that of% GFP T cells. In the case of different CD8, CD11a or CD4 targeting antibodies or nanobodies, the% DiI positive T cells and MFI in blood, liver and lung are maximal.

Part D anti-CD 7 lipid 5LNP

In this study we used LNP spiked with lipid 5 targeted CD7 (V1-PEG-DSPE conjugate) to test LNP prepared with either DMG-PEG (C14) or DPG-PEG (C16) at 2.5 or 1.5mol% of total lipid.

LNP formulations were prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa) and labeled with 0.06mol% of dic 18 (5) -DS (Invitrogen, ma). The LNP is then inserted into the targeting conjugate using the specified conditions to provide a targeted LNP formulation. The LNP was characterized as described in example 3.

Results of in vivo reprogramming of immune cells with LNP of 0.3mg/kg of lipid 5 24h after DMG-PEG or DPG-PEG (1.5%) are shown in FIGS. 63A-63T.

In the case of DMG-PEG (50%), lipid 5CD7 nanobody targeting LNP showed the largest GFP expressing T cells compared to DPG-PEG (35%), both DMG-PEG and DPG-PEG had 1.5mol% PEG-lipid. In both cases of DMG-PEG and DPG-PEG-1.5%, the other tissue livers and lungs showed equal 20% GFP expressing T cells. GFP MFI shows a similar trend. DiI positive T cells and DiI MFI showed the greatest binding only in the blood where the most GFP expression was observed.

EXAMPLE 30 in vivo reprogramming comparison of lipid 5, lipid 8, DLn-MC3-DMA LNP

In this example, the ability of CD3 (SP 34) or CD8 (V2 (nanobody)) targeted LNP with lipid 5, lipid 8 or DLn-MC3-DMA (0.1 mg/kg dose) to reprogram immune cells in vivo was tested.

LNP formulations in the following table were prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa) and labeled with 0.06mol% of dic 18 (5) -DS (Invitrogen, ma). The LNP insertion targeting conjugate is then used using the specified conditions to provide the final targeted LNP formulation. The LNP was characterized as described in example 3.

Table 39 lnp formulation

TABLE 40 analysis results of formulations

The results of in vivo reprogramming of immune cells with 0.1mg/kg of the above LNP 24h after DPG-PEG (1.5%) are shown in FIGS. 62A-62S.

Comparing DLn-MC3-DMA, lipid 8 and lipid 5 with DPG-PEG-1.5% and CD3 (hsp 34) -targeted LNP at a dose of 0.1mg/kg, lipid 5 has an advantage in reprogramming T cells and shows a maximum T cell reprogramming of about 25% with maximum GFP expression in blood and lung (blood = lung > liver > bone marrow). GFP MFI shows a similar trend. All 3 lipids showed similar DiI positive T cells, indicating that all lipids could bind equally, but lipid 5 was better in reprogramming T cells.

EXAMPLE 31 in vitro protein expression-LNP transfection of Co-cultured NK cells and T cells

This example describes the targeting of co-cultured human NK cells and T cells and their effect on transfection and translation with anti-CD 3, anti-CD 7, anti-CD 11a, anti-CD 18, anti-CD 56 (Lo Wo Tuozhu mab), anti-CD 137 (4B 4-1) and anti-CD 2 (RPA-2.10v1) Fab or nanobodies inserted into GFP mRNA DiI-labeled LNPs post-consumer.

Primary human T cells were purified using magnetic-based CD3 negative selection. 2000 ten thousand purified T cells were activated with anti-CD 3/anti-CD 28 coated beads in medium containing 100IU/mL IL-2 for 48 hours. Following activation, the activation beads were removed and the T cells were allowed to expand in medium containing 100IU/mL IL-2 for an additional 48 hours. After the expansion phase, T cells were concentrated to 100 tens of thousands of cells/mL in preparation for co-transfection with primary human NK cells.

CD3 depleted PBMCs were purified using magnetic-based CD3 positive selection and retention of negative fractions. 2000 ten thousand CD3 depleted PBMC were added to 1 well of a 6 well GREX plate for 7 days in medium containing 10ng/mL IL-15. On day 7, each well was divided into 2 parts and cells were cultured in medium containing 10ng/mL IL-15 for an additional 7 days. On day 14, NK cells were concentrated to 100 ten thousand cells/mL in preparation for co-transfection with primary human T cells.

LNP was prepared using the mixing method described in example 6, the buffer exchange method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were produced by the method described in example 4, while Nb-conjugates were produced with the difference that Nb-conjugates were separated from free Nb using a 1:1:4Nb: DSPE-5 KPEG-maleimide: DSPE-2KPEG-OCH3 and a 50kD UF membrane (Millipore Corp, biperika, ma). Using a method similar to example 12, conjugated Fab and conjugated Nb were post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye at various densities (Table 41), i.e., post-insertion into LNP containing lipid 8, GFP mRNA and DiI dye.

TABLE 41 Fab or Nb Density on post-inserted LNP surface

Targeting moiety
		αCD11a HzMHM24 bDS Fab	3,5,9g/mol
αCD18h1B4 bDS Fab	3,5,9g/mol
		αCD7 V1	3,5,9g/mol
αCD56A1 Fab bDS	3,9,19g/mol
		αCD56A2 Fab bDS	3,9,19g/mol
αCD56A3 Fab bDS	3,9,19g/mol
		αcd56 lo Wo Tuozhu mab Fab bDS	3,6,9g/mol
αCD137 4B4-1Fab bDS	3,9,18g/mol
		αCD2 9.6Fab bDS	0.75,1.5,3g/mol
αCD2 TS2/18.1Fab bDS	0.75,1.5,3g/mol
		αCD2 RPA-2.10v1 Fab bDS	0.75,1.5,3g/mol
αCD2 Lo-CD2b Fab bDS	0.75,1.5,3g/mol
		αCD2 35.1Fab bDS	0.75,1.5,3g/mol
αCD2 OKT11 Fab bDS	0.75,1.5,3g/mol
		Inactive Fab mutOKT8	9g/mol
αCD8TRX 2 NoDS (knock-out of inter-chain disulfide bond)	9g/mol
		HEPES buffered saline	Without LNP

anti-CD 56A 1 Fab sequences

A1 bDS HC(SEQ ID NO:26)：

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSNWIRQSPSGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARENIAAWTWAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

A1 bDS LC(SEQ ID NO:27)：

EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGLAPRLLIYDTSLRATDIPDRFSGSGSGTAFTLTISRLEPEDFAVYYCQQYGSSPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 56A 2 Fab sequences

A2 bDS HC(SEQ ID NO:28)：

EVQLVQSGAEVKKPGSSVKVSCKASGGTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLSSGYSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

A2 bDS LC(SEQ ID NO:29)：

DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLNWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEGEDVGDYYCMQALQSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 56A 3 Fab sequences

A3 bDS HC(SEQ ID NO:30)：

A3 bDS LC(SEQ ID NO:31)：

DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEADDVGVYYCMQSLQTPWTFGHGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 56 mab Wo Tuozhu mab Fab sequence No. Wo Tuozhu mab bDS HC (SEQ ID NO: 32):

QVQLVESGGG VVQPGRSLRL SCAASGFTFS SFGMHWVRQA

PGKGLEWVAYISSGSFTIYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARMR KGYAMDYWGQ

GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

Lo Wo Tuozhu mab bDS LC (SEQ ID NO:):

DVVMTQSPLSLPVTLGQPASISCRSSQIIIHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPHTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 2 RPA-2.10v1 Fab sequence

RPA-2.10v1 bDS HC(SEQ ID NO:34)：

EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYDMSWVRQTPEKRLEWVASISGGGFLYYLDSVKGRFTISRDNARNILYLHMTSLRSEDTAMYYCARSSYGEIMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

RPA-2.10v1 bDS LC(SEQ ID NO:35)：

DILLTQSPAILSVSPGERVSFSCRASQRIGTSIHWYQQRTTGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDVADYYCQQSHGWPFTFGGGTKLEIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 137 4B4-1 Fab sequences

4B4-1 bDS HC(SEQ ID NO:36)：

QVQLQQPGAELVKPGASVKLSCKASGYTFSSYWMHWVKQRPGQVLEWIGEINPGNGHTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARSFTTARGFAYWGQGTLVTVSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

4B4-1 bDS LC(SEQ ID NO:37)：

DIVMTQSPATQSVTPGDRVSLSCRASQTISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQDGHSFPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

On the day of LNP transfection, 5 ten thousand T cells and 5 ten thousand NK cells were added to each well of a 96 well plate in a total volume of 100. Mu.L containing 100IU/mL IL-2. mu.L of each test LNP was added to each well to facilitate simultaneous transfection of primary human T cells and NK cells at 2.5ug/mL mRNA.

24 hours after LNP transfection, the cell culture medium was aspirated from the T-cell and NK-cell co-cultures after centrifugation. T cells and NK cells were resuspended with anti-CD 3 and anti-CD 56 fluorescent labeled antibodies to facilitate independent analysis of LNP transfection in each cell type in co-culture for 20 minutes at room temperature. After incubation, cells were concentrated by centrifugation and resuspended in 1x PBS for analysis by flow cytometry. After acquisition by flow cytometry, T cells and NK cells were independently analyzed using FlowJo (flow cytometry analysis software). The frequency of GFP positive events relative to GFP negative events was calculated in cd3+ cells (T cells) or cd56+ cells (NK cells) (fig. 64A). In addition, the overall fluorescence of GFP was quantified by assessing the mean fluorescence intensity (MFI, fig. 64B). Similar frequency and MFI analyses were performed for DiI dyes (fig. 64C, fig. 64D). Together, these metrics enable quantification of LNP transfection efficiency for all targeted LNPs tested in primary human T cell and NK cell co-cultures.

Both the ex vivo expanded NK cells and T cells showed high% DiI and% GFP of LNP post-inserted non-target specific mutOKT8 Fab compared to experiments with unstimulated T cells or whole blood. Despite this difference in% frequency, there was a clear separation between mutOKT8 non-targeted LNP and many surface antigen targeted LNPs when comparing formulations (according to MFI). Consistent with previous studies, T cells were transfected with anti-CD 7, anti-CD 8, anti-CD 2, anti-CD 11a and anti-CD 18 targeted LNP, while minimal to no transfection of T cells was observed for either anti-CD 137 or anti-CD 56 targeted LNP. Similarly, NK cells can also be transfected with anti-CD 7, anti-CD 8, anti-CD 2, anti-CD 11a and anti-CD 18 targeted LNP. However, CD 56-targeted LNP with the lo Wo Tuozhu mab or A3 clone showed only highly specific transfection of NK cells compared to T cells.

This data suggests that Fab or nanobodies can achieve transfection/translation of both NK cells and T cells using anti-CD 7, anti-CD 8, anti-CD 2, anti-CD 11a or anti-CD 18 targeted LNP, while NK cells can be translated/translated with high specificity relative to other immune cells using anti-CD 56 targeted LNP.

Example 32-PEG-lipid conjugation and in vitro protein expression-CD 3-Targeted Fab with and without native interchain disulfide bonds

This example describes the conjugation of any anti-CD 3 Fab with and without native interchain disulfide bonds, purity and T cell transfection after insertion into Cy5/GFP mRNA LNP.

LNP was prepared using the mixing method described in example 6, the buffer exchange method described in example 21. Conjugates were produced using a method similar to that of example 4 except that 0.025, 0.1, 0.5mM TCEP was used for hSP DS reduction, 0.025, 0.2, 2mM TCEP was used for hSP-hlam NoDS (knock out of interchain disulfide bonds) reduction, and the conjugation reaction was performed at 37 ℃ for 2 hours prior to conjugation. SDS-PAGE was performed with 1ug of protein using manufacturer recommended conditions (Thermo, 4% -12% bis-Tris MiniGel) (FIGS. 65A, 65B). 10ul (where 1-25ug of protein was targeted) was injected and RP-HPLC (FIG. 65C, FIG. 65D) was performed using Agilent 300SB-C8 at 0.5mL/min with column temperature of 60℃and mobile phase A: water with 0.1% tfa, mobile phase B: acetonitrile with 0.1% tfa, gradient% B:0min 5%,1min 5%,6.5min 95%,8min 95%. Using a method similar to example 12, anti-CD 3 hSP34 (with and without natural interchain disulfide bonds, DS (with interchain disulfide bonds) and NoDS (without interchain disulfide bonds), see sequence below) PEG-lipid conjugated Fab was post-inserted into LNP containing lipid 8 and Cy5/GFP mRNA at various Fab densities (6, 12, 17g Fab/mol total lipid). Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. The transfection level of CD 3T cells was measured by flow cytometry.

anti-HuCD 3 hSP34-hlam Fab NoDS (without interchain disulfide bonds) and DS (with interchain disulfide bonds)

hSP34-hlam NoDS HC(SEQ ID NO:38)：

hSP34-hlam NoDS LC(SEQ ID NO:39)：

hSP34-hlam DS HC(SEQ ID NO:40)：

EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

hSP34-hlam DS LC(SEQ ID NO:41)：

QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS

The SP34 DS Fab was run on a non-reducing gel at about 49kD (fig. 65A), while SP34 NoDS Fab LC and HC were run on a non-reducing gel similarly to each other, slightly less than 28kD (fig. 65B), confirming that the interchain disulfide bond had been knocked out by mutating the corresponding cysteines in the heavy and light chains to serine. As shown in both the gel and RP-HPLC chromatograms (fig. 65C), the SP34 DS Fab reduced at different levels of TCEP showed high levels of LC/HC mono and di PEG-lipid conjugation, with the highest purity conditions being 0.025mM TCEP during reduction, while 0.1 and 0.5mM TCEP had intractable amounts of di-conjugates. In contrast, at the full range of TCEPs up to 2mM TCEP evaluated, SP34 NoDS Fab showed high purity, understating that the removal of interchain disulfide bonds had a significant impact on the ability to produce highly pure (single lipid) conjugated Fab. For T cell transfection studies, 0.025mM TCEP produced SP34 DS Fab was chosen because it was the purest reaction condition and had similar recovery to other conditions after UF purification (table 42), and 0.2mM TCEP produced SP34 NoDS Fab was chosen because it had both high purity and the best recovery after UF purification (table 43).

TABLE 42 relationship between TCEP concentration during reduction and final recovery of SP34-hlam DS conjugates after UF purification

TCEP mM	Post UF% recovery
		0.5	20.5
0.1	27.6
		0.025	24.3

TABLE 43 relationship between TCEP concentration during reduction and final recovery of SP34-hlam NoDS conjugates after UF purification

TCEP mM	Post UF% recovery
		2	14.0
0.2	55.5
		0.025	25.9

At the lowest (6 g/mol) and medium (12 g/mol) Fab densities, SP34NoDS Fab mediated higher% transfection (fig. 65E) and higher GFP expression levels (fig. 65F) than SP34 DS Fab, indicating that the potency of this conjugate was higher than that of SP34 DS Fab, consistent with its correspondingly higher purity (1 PEG-lipid per Fab, no LC-PEG-lipid).

This data suggests that knockout of the natural interchain disulfide bond achieves efficient site-specific conjugation to the C-terminal cysteine and single PEG-lipid. This broadens the range of reducing agents available to obtain high purity conjugates (1 PEG-lipid per Fab) at high process recovery rates and avoids conjugation of 2 or more PEG-lipids per Fab, which can reduce the final transfection efficiency of the targeted LNP to immune cells.

Example 33-Fab-PEG-lipid conjugation and purity-CD 2 and CD8 targeting Fab with and without native interchain disulfide bonds

This example describes the conjugation and purity of anti-CD 2 and anti-CD 8 Fab with and without native interchain disulfide bonds (fig. 47).

Conjugates were produced using a method similar to that of example 4 except that prior to conjugation, 0.025, 0.0375, 0.05, 0.0625mM TCEP was used for anti-CD 2 TS2/18.1 and 9.6DS Fab reduction, 0.05, 0.1, 0.2mM TCEP was used for anti-CD 2 TS2/18.1 and 9.6NoDS Fab (see sequences below) reduction, 0.025, 0.05, 0.1, 0.2mM TCEP was used for anti-CD 8 TRX2 NoDS Fab reduction, and the conjugation reaction was performed at 37 ℃ for 2 h. SDS-PAGE was performed with 1ug of protein using manufacturer recommended conditions (Thermo, 4% -12% bis-Tris MiniGel) (FIGS. 66A and 66B). 10ul (where 1-25ug of protein was targeted) was injected and RP-HPLC (FIG. 66C and FIG. 66D) was performed using Agilent300SB-C8 at 0.5mL/min with column temperature of 60℃and mobile phase A: water with 0.1% tfa, mobile phase B: acetonitrile with 0.1% tfa, gradient% B:0min 5%,1min 5%,6.5min 95%,8min 95%.

anti-CD 2 TS2/18.1DS Fab

TS2/18.1DS HC(SEQ ID NO:42)：

EVQLVESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

TS2/18.1DS LC(SEQ ID NO:43)：

DIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYFCQNGHNFPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

anti-CD 2.9 DS Fab

9.6DS HC(SEQ ID NO:44)：

QVQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

9.6DS LC(SEQ ID NO:45)：

NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TS2/18.1 and 9.6DS Fab were run on non-reducing gels at about 49kD (FIG. 26-1), while TS2/18.1, 9.6 and TRX2 NoDS Fab LC and HC were run on non-reducing gels similarly to each other, slightly less than 28kD (FIG. 66B), confirming that interchain disulfide bonds had been knocked out by mutating the corresponding cysteines in the heavy and light chains to serine. As shown by both SDS-PAGE (fig. 66A) and RP-HPLC chromatograms (TS 2/18.1 only, fig. 66C), TS2/18.1 and 9.6DS Fab reduced at different levels of TCEP prior to conjugation exhibited high levels of LC/HC single and double PEG-lipid conjugation, with the highest purity condition being 0.025mM TCEP during reduction, and higher TCEP levels increased the amount of LC conjugates and double conjugates (2 PEG-lipids per Fab). In contrast, at the full range of TCEPs up to 0.2mM TCEP evaluated, TS2/18.1, 9.6 and TRX2 NoDS fabs showed high purity, understating that removal of interchain disulfide bonds had a significant impact on the ability to produce highly pure (1 PEG-lipid per Fab) conjugates.

This data suggests that targeting Fab across multiple immune cells, knocking out native interchain disulfide bonds is a generalized approach for achieving efficient site-specific conjugation to C-terminal cysteines on heavy chains while avoiding conjugation to light chains and more than one PEG-lipid per Fab.

Example 34-in vitro protein expression-CD 3 and TCR targeting comparison and SP34 Fab with and without embedded disulfide bonds this example describes targeting human CD 3T cells and their effect on transfection and ifnγ secretion with anti-CD3 or anti-TCR Fab and anti-CD3 Fab with and without embedded interchain disulfide bonds (fig. 47) post-inserted into Cy5/GFP mRNA LNP.

LNP was prepared using the mixing method described in example 6, the buffer exchange method described in example 21. The conjugate was produced using a method similar to that of example 4, except that prior to conjugation, reduction was performed using 0.1mM TCEP and the conjugation reaction was performed at 37 ℃ for 2 h. Using a method similar to example 12, anti-CD 3hSP34 (with and without embedded disulfide bonds bDS and NoDS), TR66 (Bortoletto et al optimization anti-CD3 affinity for effective T cell targeting against tumor cells, eu.J of Immun.2002; frank et al Combining T cell specific activation and in vivo gene delivery through CD3-targeted lentiviral vectors, blood Adv 2020), anti-CD3 TRX4, anti-CD3 humanized UCHT1 (HzUCHT 1) and anti-CD3 telithromab PEG-lipid conjugated Fab were post-inserted into LNP containing lipid 8 and Cy5/GFP mRNA. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (clone SK 3) to distinguish between the two cell types. Ifnγ in the supernatant was measured using the manufacturer's recommended procedure (R & D Systems, DY 285B).

anti-CD 3 hSP34-hlam bDS Fab sequence

hSP34-hlam bDS HC(SEQ ID NO:46)：

EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

hSP34-hlam bDS LC(SEQ ID NO:47)：

QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAACSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAESS

anti-CD 3 TR66 bDS Fab sequence

TR66 bDS HC(SEQ ID NO:48)：

QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDNYSLDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

TR66 bDS LC(SEQ ID NO:49)：

QIVLTQSPSSLSASLGEKVTMTCRASSSVSYMNWYQQKPGTSPKRWIYDTSKVASGVPDRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 3 TRX4 bDS Fab sequences

TRX4 bDS HC(SEQ ID NO:50)：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAPGKGLEWVSTISTSGGRTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRQYSGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

TRX4 bDS LC(SEQ ID NO:51)：

DIQLTQPNSVSTSLGSTVKLSCTLSSGNIENNYVHWYQLYEGRSPTTMIYDDDKRPDGVPDRFSGSIDRSSNSAFLTIHNVAIEDEAIYFCHSYVSSFNVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAACSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS

anti-CD 3 HzUCHT1 bDS Fab sequence

HzUCHT1(Y59T)bDS HC(SEQ ID NO:52)：

EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPTKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

HzUCHT1 bDS LC(SEQ ID NO:53)：

DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 3 Tilicarbazeb antibody bDS Fab sequence Tilicarbazeb antibody bDS HC (SEQ ID NO: 54):

QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

tilicarbazemab bDS LC (SEQ ID NO: 55):

DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

the SP34 NoDS Fab mediated higher% transfection (fig. 67A) and higher GFP expression levels (fig. 67B) (quantified by Mean Fluorescence Intensity (MFI)) compared to other Fab constructs. Although SP34 bDS and TR66 bDS Fab mediated similar levels of% transfection (in terms of GFP), the expression level was lower than that of SP34 NoDS Fab (quantified by mean fluorescence intensity). For SP34, noDS and bDS Fab forms had similar levels of ifnγ secretion (fig. 67C). In addition, while TR66, TRX4, hzUCHT1, and telizumab had higher levels of ifnγ secretion than SP34, they exhibited lower levels of GFP expression (fig. 67B).

This data suggests that Fab targeting across a variety of CD3, whether they have kappa or lambda light chains, many are capable of mediating high transfection/translation in NoDS or bDS, exemplifying CD3 as a robust T cell target for mediating CD8 and CD 4T cell transfection and translation. For the SP34 clone, the NoDS format was superior to the bDS format in terms of T cell transfection/translation efficiency. In addition, this data suggests that T cell activation does not guarantee efficient transfection and translation.

Example 35 in vitro protein expression-CD 8-targeted Fab with and without embedded disulfide bonds and other CD 2-targeted Fab clones

This example describes targeting of human CD8T cells with anti-CD 8 Fab or anti-CD 2 Fab in the form of NoDS or bDS inserted into Cy5/GFP mRNA LNP after use and their effect on transfection and translation.

LNP was prepared using the mixing method described in example 6, the buffer exchange method described in example 21. Fab-lipid conjugates were generated from the method described in example 4. Using a method similar to example 12, anti-CD 3 hSP, anti-CD 8TRX2 and anti-CD 2 clones Lo-CD2b (ATCC, PTA-802), 35.1 (ATCC, HB-222) and OKT11 (ATCC, CRL-8027) PEG-lipid conjugated Fab were post-inserted into LNP containing lipid 8 and Cy5/GFP mRNA. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. The transfection level of CD8 cells was measured by flow cytometry.

anti-CD 8TRX2 bDS Fab sequence

TRX2 bDS HC(SEQ ID NO:56)：

QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

TRX2 bDS LC(SEQ ID NO:57)：

DIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 2 Lo-CD2b bDS Fab sequence

Lo-CD2b bDS HC(SEQ ID NO:58)：

EVQLVESGGGLVQPGASLKLSCVASGFTFSDYWMSWVRQTPGKPMEWIGHIKYDGSYTNYAPSLKNRFTISRDNAKTTLYLQMSNVRSEDSATYYCAREAPGAASYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

Lo-CD2b bDS LC(SEQ ID NO:59)：

DVVLTQTPVAQPVTLGDQASISCRSSQSLVHSNGNTYLEWFLQKPGQSPQLLIYKVSNRFSGVPDRFIGSGSGSDFTLKISRVEPEDWGVYYCFQGTHDPYTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 2.1 bDS Fab sequence

35.1bDS HC(SEQ ID NO:60)：

EVQLQQSGAELVKPGASVKLSCRTSGFNIKDTYIHWVKQRPEQGLKWIGRIDPANGNTKYDPKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCVTYAYDGNWYFDVWGAGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

35.1bDS LC(SEQ ID NO:61)：

DIKMTQSPSSMYVSLGERVTITCKASQDINSFLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMEIYYCLQYDEFPYTFGGGTKLEMKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 2 OKT11 bDS Fab sequences

OKT11 bDS HC(SEQ ID NO:62)：

QVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

OKT11 bDS LC(SEQ ID NO:63)：

DIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

The interchain disulfide knockout (NoDS) anti-CD 8TRX2 Fab had slightly higher% transfection (fig. 68A) and GFP expression levels (fig. 68B) than the disulfide embedded (bDS) TRX2 Fab, but with little difference. Similar to example 17, none of the CD2 targeted fabs explored herein performed as well as anti-CD 3 or anti-CD 8 fabs.

This data shows that TRX2 clones in NoDS form are preferred, but Fab in bDS form can mediate efficient T cell transfection/translation. In addition, CD8 and CD3 are preferred targets over CD2 for the clones evaluated.

Example 36 in vitro protein expression-CD 8-Targeted Fab with and without embedded disulfide bonds and other CD 2-Targeted Fab clones

This example describes targeting human T cells and their effect on transfection/translation and ifnγ cytokine secretion by co-targeting with anti-CD 3 and anti-CD 11a or anti-CD 3 and anti-CD 18 Fab post-inserted into Cy5/GFP mRNA LNP.

LNP was prepared using the mixing method described in example 6, the buffer exchange method described in example 21. Fab-lipid conjugates were generated from the method described in example 4. Using a method similar to example 12, anti-CD 3 hSP34, anti-CD 11. Alpha. HzMHM24, anti-CD 18 Erlizumab (Erlizumab) PEG-lipid conjugated Fab was post-inserted into LNP containing lipid 8 and Cy5/GFP mRNA. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 7) to distinguish between the two cell types. Ifnγ in the supernatant was measured using the manufacturer's recommended procedure (R & D Systems, DY 285B).

Targeting CD11a or CD18 alone mediated transfection of both CD8 and CD 4T (fig. 69A) and GFP expression (fig. 69B). Importantly, by co-targeting the anti-CD 3 clone with any anti-CD 11a or CD18 clone, ifnγ secretion levels were reduced by nearly 50%, while transfection and translation levels were similar to or higher than that of CD3 targeted alone (fig. 69C).

This data suggests that targeting CD11a and CD18 can mediate efficient immune cell transfection/translation, and co-targeting CD3 with CD11a or CD18 can significantly reduce cytokine release by T cells without negatively impacting T cell transfection and protein translation. Another anti-CD 3 clone in the NoDS Fab form could approach similar levels of transfection/translation of SP34 in the NoDS Fab form.

anti-CD 11a HzMHM24 bDS Fab sequence

HzMHM24 bDS HC(SEQ ID NO:64)：

EVQLVESGGGLVQPGGSLRLSCAASGYSFTGHWMNWVRQAPGKGLEWVGMIHPSDSETRYNQKFKDRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARGIYFYGTTYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

HzMHM24 bDS LC(SEQ ID NO:65)：

DIQMTQSPSSLSASVGDRVTITCRASKTISKYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 18 h1B4 bDS Fab sequence

h1B4 bDS HC(SEQ ID NO:66)：

EVQLVESGGDLVQPGRSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVAAIDNDGGSISYPDTVKGRFTISRDNAKNSLYLQMNSLRVEDTALYYCARQGRLRRDYFDYWGQGTLVTVSTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

h1B4 bDS LC(SEQ ID NO:67)：

DIQMTQSPSSLSASVGDRVTITCRASESVDSYGNSFMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 18 erlizumab bDS Fab sequence erlizumab bDS HC (SEQ ID NO: 68):

EVQLVESGGGLVQPGGSLRLSCATSGYTFTEYTMHWMRQAPGKGLEWVAGINPKNGGTSHNQRFMDRFTISVDKSTSTAYMQMNSLRAEDTAVYYCARWRGLNYGFDVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

erlizumab bDS LC (SEQ ID NO: 69):

DIQMTQSPSSLSASVGDRVTITCRASQDINNYLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

example 37 in vitro protein expression Co-targeting LNP with CD4 and CD8 Fab or CD4/CD8 Fab-ScFv bispecific

This example describes the targeting of human T cells with CD4 Fab with CD8 ScFv (Fab-ScFv) distal to the CD4 Fab light chain, and their effect on transfection and ifnγ secretion with anti-CD 4 or anti-CD 8 Fab, anti-CD 4 and anti-CD 8 Fab inserted into Cy5/GFP mRNA LNP.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were generated from the method described in example 4. Using a method similar to example 12, anti-CD 3 hSP, anti-CD 4 ibazumab, anti-CD 8 TRX2 conjugated Fab and CD4/CD8 ibazumab/TRX 2 Fab-ScFv were post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types. Ifnγ in the supernatant was measured using the manufacturer's recommended procedure (R & D Systems, DY 285B).

Post-insertion of both anti-CD 8 and anti-CD 4 Fab together showed similar CD8 and CD 4T cell transfection and protein expression relative to Fab post-insertion alone (fig. 70A and 70B). The CD4/CD8 Fab-ScFv bispecific showed slightly reduced CD4 and CD 8T cell transfection compared to the post-insertion of CD4 and CD8 Fab together. In contrast to targeting CD3 with SP34 Fab, none of the CD4, CD8 or CD4/CD8 co-targeting conditions mediated significant ifnγ release (fig. 70C).

This data suggests that dual specificity targeting moieties can be leveraged to minimize loss of targeting function for a single protein construct targeting 2 different immune cell types as compared to insertion of the targeting moiety alone into the same LNP.

anti-CD 4/CD8 ibuzumab/TRX 2 bDS Fab-ScFv sequences

Abamectin/TRX 2 bDS Fab-ScFv HC (SEQ ID NO: 70):

QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKT

HTCHHHHHH

Abamectin/TRX 2 bDS Fab-ScFv LC (SEQ ID NO: 71):

DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGESGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIK

example 38 in vitro protein expression-CD 4-Targeted Fab without native inter-chain disulfide bonds or with embedded inter-chain disulfide bonds

This example describes targeting human T cells with anti-CD 3 or anti-CD 4 Fab inserted into Cy5/GFP mRNA LNP and their effect on transfection and ifnγ secretion.

LNP was prepared using the mixing method described in example 6, the buffer exchange method described in example 21. Fab-lipid conjugates were generated from the method described in example 4. Using a method similar to example 12, anti-CD 3 hSP, anti-CD 4 ibalizumab, anti-CD 4 humanized OKT4 PEG-lipid conjugated Fab and Nb were post-inserted into LNP containing lipid 8 and Cy5/GFP mRNA. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 4) to distinguish between the two cell types. Ifnγ in the supernatant was measured using the manufacturer's recommended procedure (R & D Systems, DY 285B).

In CD 4-targeted Fab, ibalizumab mediated higher% transfection (fig. 71A) and GFP expression levels (fig. 71B) (quantified by Mean Fluorescence Intensity (MFI)), however it was lower than anti-CD 3 SP34 Fab. None of the anti-CD 4 fabs mediated significant ifnγ secretion levels, while the anti-CD 3 SP34 fabs exhibited higher levels of ifnγ compared to the non-targeted mutOKT8 fabs (fig. 71C).

This data suggests that anti-CD 4 Fab without native interchain disulfide bonds (ibalizumab, noDS) or with embedded interchain disulfide bonds (OKT 4, bDS) can mediate highly specific LNP transfection and protein translation of cd4+ T cells, and targeting CD4 can avoid T cell activation and ifnγ release, as compared to cd8+ T cells.

anti-CD 4 ibalizumab NoDS Fab sequence ibalizumab NoDS LC (SEQ ID NO: 72):

abamectin NoDS HC (SEQ ID NO: 73):

anti-CD 4 OKT4 bDS Fab sequences

OKT4 bDS LC(SEQ ID NO:74)：

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKRLEWVSAISDHSTNTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARKYGGDYDPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

OKT4 bDS HC(SEQ ID NO:75)：

DIQMTQSPSSLSASVGDRVTITCQASQDINNYIAWYQHKPGKGPKLLIHYTSTLQPGIPSRFSGSGSGRDYTLTISSLQPEDFATYYCLQYDNLLFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

Example 39 in vitro protein expression-other CD4 targeting Fab clones and CD4 targeting nanobodies

This example describes the targeting of human T cells with anti-CD 3 or anti-CD 4 Fab inserted post-GFP mRNA DiI-tagged LNP and their effect on transfection and ifnγ secretion.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were produced by the method described in example 4, while Nb-conjugates were produced with the difference that Nb-conjugates were separated from free Nb using 1:1:4Nb: DSPE-3.4 KPEG-maleimide: DSPE-2KPEG-OCH3 and a 50kD UF membrane (Millipore Corp, biperika, ma). Using a method similar to example 12, anti-CD 3 hSP, anti-CD 4 ibalizumab, anti-CD 4 hBF conjugated Fab and conjugated Nb (derived from llama immunization) were post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (OKT 3) to distinguish between the two cell types. Ifnγ in the supernatant was measured using the manufacturer's recommended procedure (R & D Systems, DY 285B).

In the CD4 targeting conjugate, anti-CD 4 mediated slightly higher% transfection (fig. 72A) and GFP expression levels (fig. 72B) (quantified by Mean Fluorescence Intensity (MFI)), however it was lower than anti-CD 3 SP34 Fab. For CD 4-targeted Fab and Nb, transfection and translation were only observed in the cd4+ T cell population. None of the anti-CD 4 fabs mediated significant ifnγ secretion levels, while the anti-CD 3 SP34 fabs exhibited higher levels of ifnγ compared to the non-targeted mutOKT8 fabs.

This data suggests that both Fab and nanobodies can mediate highly specific LNP transfection and protein translation of cd4+ T cells, and targeting CD4 can avoid T cell activation and ifnγ release, as compared to CD 8T cells.

anti-CD 4T 023200008Nb sequence (SEQ ID NO: 76)

CDR1, CDR2, CDR3 are underlined, based on IMGT name:

EVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSSTGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNPARWDGYDFRGQGTLVTVSSGGCGGHHHHHH

EXAMPLE 40 in vitro protein expression-CD 8 targeting nanobody cloning

This example describes the targeting of human T cells with anti-CD 3, anti-CD 8 Fab or anti-CD 8 nanobodies inserted post-GFP mRNA DiI-labeled LNP and their effect on transfection and ifnγ secretion.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were produced by the method described in example 4, while Nb-conjugates were produced with the difference that Nb-conjugates were separated from free Nb using a 1:1:4Nb: DSPE-5 KPEG-maleimide: DSPE-2KPEG-OCH3 and a 50kD UF membrane (Millipore Corp, biperika, ma). Using a method similar to example 12, conjugated Fab and conjugated Nb (derived from llama or alpaca immunization) were post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types. Ifnγ in the supernatant was measured using the manufacturer's recommended procedure (R & D Systems, DY 285B).

CD 8-targeted Nb conjugates exhibited higher% transfection (fig. 73A) and GFP expression levels (fig. 73B) compared to anti-CD 8 TRX 2. For CD 8-targeted Nb, transfection and translation were observed only in the CD 8T cell population relative to mutOKT8 Fab. anti-CD 8 Nb did not mediate significant ifnγ secretion levels compared to non-targeted mutOKT8 Fab, whereas anti-CD 3 SP34 Fab exhibited higher levels of ifnγ (fig. 73C).

This data suggests that both Fab and nanobodies can mediate highly specific LNP transfection and protein translation of CD 8T cells, and targeting CD8 can avoid T cell activation and ifnγ release, as compared to CD 4T cells.

anti-CD 8 BDSn Nb sequence (SEQ ID NO: 77)

CDR1, CDR2, CDR3 are underlined, based on IMGT name:

EVQLVESGGGLVQAGGSLRLSCAASGSTFSDYGVGWFRQAPGKGREFVADIDWNGEHTSYADSVKGRFATSRDNAKNTAYLQMNSLKPEDTAVYYCAADALPYTVRKYNYWGQGTQVTVSSGGCGGHHHHHH

example 41 in vitro protein expression-CD 3 and CD7 targeting nanobodies with 2K or 5K PEG

This example describes the targeting of human T cells with anti-CD 3, anti-CD 7 Fab or anti-CD 8 nanobodies inserted post-GFP mRNA DiI-labeled LNP and their effect on transfection and ifnγ secretion.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were produced by the method described in example 4, while Nb-conjugates were produced with the difference that Nb-conjugates were separated from free Nb using 1:1:4Nb: DSPE-5 KPEG-maleimide or DSPE-3.4 KPEG-maleimide: DSPE-2KPEG-OCH3 and 50kD UF membranes (Millipore Corp, biperika, ma, usa). Conjugated Fab and conjugated Nb (E11 and G03) V1 (anti-CD 7) were post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye using a method similar to example 12. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types.

For both anti-CD 3 Nb clones and anti-CD 7 Nb, longer 5K PEG improved% transfection (fig. 74A) and GFP expression levels (fig. 74B) compared to 2K PEG. The difference between the conjugate PEG lengths was more pronounced for anti-CD 3 Nb than for anti-CD 7 Nb.

This data suggests that nanobody conjugates can benefit from PEG lengths exceeding 2K, and that different clones can have different degrees of improvement.

anti-CD 3T 0170117G03-A Nb sequence (SEQ ID NO: 78)

EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNIMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSAGGGSGGHHHHHHC

anti-CD 3T 0170060E11 Nb sequence (SEQ ID NO: 79)

EVQLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRDLVAVIGSRGNNRGRTNYADSVKGRFTISRDGTGNTVYLLMNKLRPEDTAIYYCNTAPLVAGRPWGRGTLVTVSSGGGSGGHHHHHHC

anti-CD 7V 1 Nb sequence (SEQ ID NO: 80)

DVQLQESGGGLVQAGGSLRLSCAVSGYPYSSYCMGWFRQAPGKEREGVAAIDSDGRTRYADSVKGRFTISQDNAKNTLYLQMNRMKPEDTAMYYCAARFGPMGCVDLSTLSFGHWGQGTQVTVSITGGGCHHHHHHHH

Example 42 in vitro protein expression-CD 8, CD3, CD28, CD4 and TCR targeting nanobody with 2K or 5K PEG

This example describes the targeting of human T cells with anti-CD 8, anti-CD 3, anti-CD 4 Fab, anti-CD 8, anti-CD 3, anti-CD 28, anti-CD 4, and anti-TCR nanobodies post-insertion into GFP mRNA DiI-labeled LNP and their effect on transfection and ifnγ secretion.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were produced by the method described in example 4, while Nb-conjugates were produced with the difference that either 1:1:4Nb: DSPE-2 KPEG-maleimide: DSPE-2KPEG-OCH3 or Nb: DSPE-5 KPEG-maleimide: DSPE-2KPEG-OCH3 and 50kD UF membrane (Millipore Corp, biperika, ma) were used to separate Nb-conjugates from unconjugated Nb. Using a method similar to example 12, conjugated Fab and conjugated Nb (derived from llama or alpaca immunization) were post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types.

For all targets evaluated, nanobodies conjugated to longer 5K PEG improved% transfection (fig. 75A and 75B) and GFP expression levels (fig. 75C and 75D) overall compared to 2K PEG, except that anti-CD 8 clone E05 showed the opposite relationship. The magnitude of the improvement appears to be clone specific.

This data shows that the preferred PEG length of nanobody PEG-lipid conjugates is greater than 2K regardless of the target.

anti-TCR T017000700Nb sequence (SEQ ID NO: 81)

CDR1, CDR2, CDR3 are underlined, based on IMGT name:

EVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGCGGHHHHHH

anti-CD 28CD065G01 Nb sequence (SEQ ID NO: 82)

EVQLVESGGGLVQPGGSLRLSCAASGSIFRLHTMEWYRRTPETQREWVATITSGGTTNYPDSVKGRFTISRDDTKKTVYLQMNSLKPEDTAVYYCHAVATEDAGFPPSNYWGQGTLVTVSSGGCGGHHHHHH

anti-CD 3T 0170061C09 Nb sequence (SEQ ID NO: 83)

EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGREREFVAAIVWSDGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGCGGHHHHHH

anti-CD 4T 023200008Nb sequence (SEQ ID NO: 76)

Example 43 in vitro protein expression-CD 8, CD7 and CD3 targeting nanobodies with 5K or 3.4K PEG

This example describes the targeting of human T cells with anti-CD 8, anti-CD 3 Fab and anti-CD 8, anti-CD 7 and anti-CD 3 nanobodies post-insertion into GFP mRNA DiI-labeled LNP and their effect on transfection.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were produced by the method described in example 4, while Nb-conjugates were produced with the difference that 1:1:4Nb: DSPE-3.4 KPEG-maleimide: DSPE-2KPEG-OCH was used ₃ Or Nb is DSPE-5 KPEG-maleimide, DSPE-2KPEG-OCH ₃ And a 50kD UF membrane (Millipore Corp, biperi, ma) separates Nb-conjugate from unconjugated Nb. Using a method similar to example 12, conjugated Fab and conjugated Nb (derived from llama or alpaca immunization) were post-inserted into LNP containing lipid 5 and GFP mRNA with DiI dye at a temperature of 37℃for 4h. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types.

For all targets evaluated, nb conjugated with shorter 3.4K PEG was similar to longer 5K PEG in% transfection (fig. 76A) and GFP expression levels (fig. 76B), if not slightly better, or in the case of anti-CD 7V 1 Nb, 3.4K PEG mediated higher transfection and expression than 5K PEG.

This data shows that the generally preferred PEG length of nanobody-PEG-lipid conjugates is 3.4K PEG compared to shorter 2K PEG as previously described in example 42 or longer 5K PEG as described herein, regardless of target.

Example 44 in vitro protein expression-2K and 3.4K PEG spacer for Fab

This example describes anti-CD 3, anti-CD 4, anti-CD 8, anti-CD 28 Fab targeted human T cells and their effect on transfection in LNP labeled with post-insertion GFP mRNA DiI.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were generated from the method described in example 4. Using a method similar to example 12, conjugated Fab (12D 2) was post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types.

While most Fab clones did not differ in% transfection (fig. 77A) and GFP expression levels (fig. 77B) between 2 PEG lengths, anti-CD 4 ibazumab showed an increase in transfection efficiency from 2K PEG to 3.4K PEG, whereas from 2K PEG to 3.4K PEG, anti-CD 3 SP34 transfection efficiency was reduced.

This data shows that, in general, for Fab-PEG-lipid conjugates, a 2K PEG spacer is preferred, however some clones may benefit from longer PEG spacers. In addition, it was shown that anti-CD 3 clone 12D2 with embedded disulfide bonds with 2K or 3.4K PEG could efficiently transfect both CD8 and a subset of CD 4T cells.

anti-CD 3 12D2 bDS Fab sequence

12D2 bDS HC(SEQ ID NO:84)：

EVKLVESGGGLVQPGRSLRLSCAASGFNFYAYWMGWVRQAPGKGLEWIGEIKKDGTTINYTPSLKDRFTISRDNAQNTLYLQMTKLGSEDTALYYCAREERDGYFDYWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

12D2 bDS LC(SEQ ID NO:85)：

QFVLTQPNSVSTNLGSTVKLSCKRSTGNIGSNYVNWYQQHEGRSPTTMIYRDDKRPDGVPDRFSGSIDRSSNSALLTINNVQTEDEADYFCQSYSSGIVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAACSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAESS

EXAMPLE 45 in vitro protein expression-CD 8 targeting nanobody mRNA titration

This example describes the targeting of human T cells with anti-CD 3, anti-CD 8 Fab or anti-CD 8 nanobodies post-inserted into GFP mRNA DiI-tagged LNPs and their effect on transfection and protein expression.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were produced by the method described in example 4, while Nb-conjugates were produced with the difference that Nb-conjugates were separated from free Nb using 1:1:4Nb: DSPE-3.4 KPEG-maleimide: DSPE-2KPEG-OCH3 and a 50kD UF membrane (Millipore Corp, biperika, ma). Using a method similar to example 12, conjugated Fab and conjugated Nb (derived from alpaca immunization) were post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye. Transfection with human CD 3T cells was performed at approximately 2.5, 0.5 and 0.1. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types.

Down to 0.1ug/mL mRNA, CD8 targeted Nb conjugate showed% transfection (fig. 78A) and GFP expression levels (fig. 78B) greater than mutOKT8 negative control.

This data shows that nanobodies conjugated to 3.4K PEG-lipids can mediate efficient T cell transfection at low levels of mRNA concentration in solution.

EXAMPLE 46 in vitro protein expression-CD 28 targeting Fab clones

This example describes anti-CD 28, anti-CD 8, anti-CD 4, anti-CD 3 Fab targeted human T cells post-consumer insertion into GFP mRNA LNP (doped with DiI dye) and their effect on transfection and ifnγ secretion.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were generated from a similar procedure as described in example 4. Using a method similar to example 12, anti-CD 28G 8A, anti-CD 28E 12, anti-CD 28 CD28.9.3, anti-CD 28 HzTN228, anti-CD 28 TGN2122.C/H.

PEG-lipid conjugated Fab was post-inserted into LNP containing lipid 8 and GFP mRNA and doped with DiI dye. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types. Ifnγ in the supernatant was measured using the manufacturer's recommended procedure (R & D Systems, DY 285B).

Although most CD 28-targeted Fab showed greater than the CD8 and CD 4T cell GFP transfection (fig. 79A) and expression levels (fig. 79B) of the inserted particles after mutOKT8, none of the clones evaluated exceeded targeting a single T cell subset with anti-CD 4 hBF, anti-CD 8 TRX2 or targeting both subsets with anti-CD 3 SP 34. None of the clones evaluated elicited significant ifnγ secretion compared to mutOKT8LNP except SP34 (fig. 79C).

This data shows that although both CD4 and a subset of CD 8T cells can be transfected, targeting CD28 has no advantage in transfection/translation efficiency over targeting CD4, CD8 or CD3 for the clones evaluated.

anti-CD 28G 8A Fab sequence

8G8A bDS HC(SEQ ID NO:86)：

EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSLTSEDSALYCARWGDGNYWGRGTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

8G8A bDS LC(SEQ ID NO:87)：

DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISMEAEDAATYFCHQYHRSPTFGGGTKLETKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 28 2E12 Fab sequences

2E12 bDS HC(SEQ ID NO:88)：

QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

2E12 bDS LC(SEQ ID NO:89)：

DIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 28 CD28.9.3Fab sequences

CD28.9.3bDS HC(SEQ ID NO:90)：

QVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

CD28.9.3bDS LC(SEQ ID NO:91)：

DIVLTQSPAS LAVSLGQRAT ISCRASESVEYYVTSLMQWY QQKPGQPPKL

LIFAASNVES GVPARFSGSG SGTNFSLNIHPVDEDDVAMY FCQQSRKVPY

TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 28 HzTN228 Fab sequence

HzTN228 bDS HC(SEQ ID NO:92)：

QVQLQESGPGLVKPSETLSLTCAVSGFSLTSYGVHWIRQPGKGLEWLGVIWPGTNFNSALMSRLTISEDTSKNQVSLKLSSVTAADTAVYCARDRAYGNYLYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

HzTN228 bDS LC(SEQ ID NO:93)：

DIQMTQSPSLSASVGDRVTITCRASESVEYVTSLMQWYQKPGKAPKLLIYAASNVDSGVPSRFSGSGTDFTLTISLQPEDIATYCQSRKVPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 28 TGN2122.C Fab sequences

TGN2122.C bDS HC(SEQ ID NO:94)：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYKIHWVRQAPGQGLEWIGYIYPYSGSSDYNQKFKSRATLTVDNSISTAYMELSRLRSDDTAVYYCARGGDAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

TGN2122.C bDS LC(SEQ ID NO:95)：

DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQRKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDYTLTISSLQPEDFATYFCQNILGTWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

anti-CD 28 TGN2122.H Fab sequences

TGN2122.H bDS HC(SEQ ID NO:96)：

EVQLVESGGGLVQPGGSLRLSCAASGFTFNIYYMSWVRQAPGKGLELVAAINPDGGNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARYGGPGFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

TGN2122.H bDS LC(SEQ ID NO:97)：

ENVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLWIYDTSKLASGIPARFSGSGSRNDYTLTISSLEPEDFAVYYCFPGSGFPFMYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

Example 47 in vitro protein expression-CD 3, CD4, CD7, CD8, CD11a, CD18, CD28 and TCR targeting Fab and nanobody

This example describes anti-CD 3, anti-CD 4, anti-CD 7, anti-CD 8, anti-CD 11a, anti-CD 18, anti-CD 28 and anti-TCR Fab and Nb targeted human T cells and their effects on transfection and ifnγ secretion in LNP labeled with post-insertion GFP mRNA DiI.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were produced by the method described in example 4, while Nb-conjugates were produced with the difference that Nb-conjugates were separated from free Nb using 1:1:4Nb: DSPE-3.4 KPEG-maleimide: DSPE-2KPEG-OCH3 and a 50kD UF membrane (Millipore Corp, biperika, ma). Using a method similar to example 12, conjugated Fab and conjugated Nb were post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL, 0.5, ug/mL, and 0.1ug/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types.

All clones evaluated mediated a certain level of transfection (fig. 80A, 80B) and GFP expression levels (fig. 80C, 80D) relative to the mutOKT8 LNP control. For simultaneous targeting of both CD8 and CD 4T cell subsets, anti-CD 3 and anti-CD 7 are excellent by having the highest transfection/translation between the two cell subsets at the highest and second highest mRNA doses. anti-TCR clones had high transfection/translation efficiency at the highest dose, but declined at the second highest dose. For a particular subset of T cells targeting, either Fab or Nb is used, targeting anti-CD 8 or anti-CD 4 provides the highest specificity for its respective subset. Targeting CD3 or TCR elicited T cell activation and ifnγ secretion, whereas other targeted clones did not elicit levels significantly exceeding mutOKT8 LNP (fig. 80E).

This data suggests that Fab or Nb targeting of CD3 or CD7 is preferred to achieve high transfection of both CD4 and CD 8T cell subsets. For targeting a subset of CD4 or CD 8T cells individually, subset-specific anti-CD 4 or anti-CD 8Fab or Nb is preferably used to achieve high transfection of their respective T cell subsets. Targeting CD3 or TCR may elicit ifnγ secretion, while targeting CD4, CD7, CD8, CD11a, anti-CD 18 or anti-CD 28 may avoid ifnγ secretion.

EXAMPLE 48 in vitro protein expression-Co-targeting of CD7 and CD8 LNPs

This example describes the anti-CD 7 anti-CD 8 Nb targeting human T cells and their effect on transfection and ifnγ secretion with GFP mRNA DiI-tagged LNP inserted either alone or together.

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were produced by the method described in example 4, while Nb-conjugates were produced with the difference that Nb-conjugates were separated from free Nb using a 1:1:4Nb: DSPE-5 KPEG-maleimide: DSPE-2KPEG-OCH3 and a 50kD UF membrane (Millipore Corp, biperika, ma). Using a method similar to example 12, conjugated Fab and conjugated Nb were post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types. Ifnγ in the supernatant was measured using the manufacturer's recommended procedure (R & D Systems, DY 285B).

For both anti-CD 8 Nb clones combined with anti-CD 7V 1 Nb (V3 and V4), the% transfection (fig. 81A) and GFP expression levels in the CD8T cell subset (fig. 81B) were higher than for CD8 targeting with Nb alone or CD7 targeting with TRX2 NoDS Fab targeting. For CD8T cells, the CD7/CD8 targeting combination approaches similar GFP expression levels as anti-CD 3 SP34NoDS Fab, while maintaining similar (if not lower) transfection levels in CD 4T cells. Although CD8/CD7 co-targeting achieved similar transfection/translation levels in the CD8T cell population as those of anti-CD 3 Fab, the amount of ifnγ secreted by T cells was not significant relative to the nonspecific mutOKT8 control LNP (fig. 81C).

This data suggests that co-targeting CD7 and CD8 can mediate efficient transfection in the CD8T cell population while avoiding substantial ifnγ secretion.

Example 49 in vitro protein expression-CD 7 and CD8 bispecific targeting LNP and CD8 targeting ScFv

This example describes the targeting of human T cells and their effect on transfection/translation and ifnγ secretion with anti-CD 8TRX2 Fab NoDS or anti-CD 8TRX2 ScFv alone or together post-inserted into GFP mRNA DiI-tagged LNP and the bispecific design described in figure 47 post-inserted into GFP mRNA DiI-tagged LNP (including anti-CD 7/anti-CD 8 2 xhh (V1/V2), anti-CD 8/anti-CD 7 2 xhh (V2/V1) or anti-CD 7/anti-CD 8 VHH-CH1/VHH-Vk bDS).

LNP was prepared using the microfluidic mixing method described in example 6 and the discontinuous diafiltration method described in example 25. LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa), lipid 8 as an ionizable lipid, and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma, usa). Fab-lipid conjugates were produced by the method described in example 4, while ScFv or Nb conjugates were produced with the difference that ScFv or Nb-conjugates were separated from free proteins using a 1:1:4nb:dspe-3.4 KPEG-maleimide DSPE-2KPEG-OCH3 and a 50kD UF membrane (Millipore Corp, biperika, ma). Using a method similar to example 12, conjugated Fab, conjugated ScFv and conjugated Nb were post-inserted into LNP containing lipid 8 and GFP mRNA with DiI dye. Transfection with human CD 3T cells was performed at approximately 2.5. Mu.g/mL mRNA for approximately 24h. Transfection levels of both CD8 and CD4 cells were measured by flow cytometry using an anti-CD 4 antibody (SK 3) to distinguish between the two cell types. Ifnγ in the supernatant was measured using the manufacturer's recommended procedure (R & D Systems, DY 285B).

TRX2 ScFv mediated slightly reduced% transfection (fig. 82A) and GFP expression levels (fig. 82B) in the CD 8T cell subset relative to anti-CD 8 TRX2 NoDS Fab, whereas the signal was greater than non-targeted mutOKT8 Fab LNP. Co-targeting of CD8 and CD7 LNPs with post-inserted anti-CD 8 and anti-CD 7 Nb or post-inserted dual-idiosyncratic agents (including anti-CD 7/anti-CD 82 xVHH, anti-CD 8/anti-CD 7 2xVHH and anti-CD 7/anti-CD 8 VHH-CH1/VHH-Vk bDS) all showed high levels of% CD 8T cell transfection and higher levels of GFP expression compared to anti-CD 3 SP34-hlam NoDS Fab, indicating that combined Nb has a synergistic effect on CD8 and CD7 targeting similar to the observations with Fab co-targeting of CD8 (clone TRX 2) and CD7 (clone TH-69) in example 17. Although CD7/CD8 co-targeting achieved similar or better transfection/translation than that of anti-CD 3 SP34 NoDS Fab in the CD 8T cell subset, the amount of ifnγ secreted by T cells was not significant relative to the nonspecific mutOKT8 control LNP and compared to SP34 (fig. 82C).

This data indicates that ScFv alone is capable of mediating transfection efficiencies similar to those of Fab. In addition, it shows that when targeting both CD7 and CD8 on the same LNP, a synergistic effect on transfection/translation can be achieved, whether the targeting moiety is post-inserted as separate proteins together or post-inserted as a dual-targeting dual-specific foreign body.

anti-CD 8 TRX2 ScFv sequence (SEQ ID NO: 98):

QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIKGGGSGGCGGHHHHHH

V1 VHH-CH1 bDS HC(SEQ ID NO:99)：

DVQLQESGGGLVQAGGSLRLSCAVSGYPYSSYCMGWFRQAPGKEREGVAAIDSDGRTRYADSVKGRFTISQDNAKNTLYLQMNRMKPEDTAMYYCAARFGPMGCVDLSTLSFGHWGQGTQVTVSITASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

example 50-lipid 2, lipid 6, lipid 12 and lipid 13LNP Properties and in vitro GFP protein expression in Primary human T cells

This example compares the properties of LNP prepared using lipid 2, lipid 6, lipid 12 and lipid 13 and GFP protein expression in primary human T cells in vitro. LNP formulations were prepared using a microfluidic mixing method (described in example 6) and using a discontinuous diafiltration method for ethanol removal (described in example 25). LNP was formulated using mRNA encoding eGFP (TriLink Biotechnologies, california, usa) and labeled with 0.01mol% dic 18 (5) -DS (Invitrogen, ma), using the lipid ratios shown in formulation table 44 below. The LNP insertion targeting conjugate is then used using the specified conditions to provide the final targeted LNP formulation. The LNP was characterized as described in example 3.

TABLE 44 LNP formulation composition and antibody insertion conditions

TABLE 45 LNP size, charge (dynamic light scattering) and mRNA encapsulation (Ribogreen assay)

Lipid 2, lipid 6, lipid 12 and lipid 13 were formulated with 1.5 mole% DPG-PEG, as seen in tables 44 and 45, all LNPs showed hydrodynamic Diameters (DLS) below 100nm in pH 7.4HEPES buffer. Buffer exchange to pH 6.5MES neutralising antibody insertion resulted in an increase in size and polydispersity of all four lipid compositions. However, lipid 2 and lipid 6LNP showed significantly greater changes in size distribution compared to lipid 12 and lipid 13LNP (fig. 83A and 83B). As seen in fig. 83C, lipid 2 and lipid 6 showed greater positive surface charges relative to lipids 12 and 13 under both physiological and acidic pH conditions (pH 7.4 and pH 5.5), indicating that LNP apparent pK due to mono-and di-hydroxyethyl substitution of the ionizable amine head groups, respectively _a The transition to lower values was significant (in lipids 12 and 13 LNP). In addition, low levels of dye accessible mRNA were observed in all four LNP compositions<20%) and good RNA encapsulation efficiency (in the parental LNP sample>80% mRNA) (Table 45 and FIG. 83D). The resulting targeted LNP was evaluated in primary human T cells using the in vitro transfection protocol described in example 8. As seen in fig. 84E, at all LNP doses tested, all formulations were well tolerated by T cells (T cell viability remained similar to PBS control). As illustrated by dii+ and DiI MFI values (fig. 84C and 84D), all formulations showed similar levels of cell association at most of the dose levels tested, indicating that the conjugate insertion process was independent of the chemical composition of the ionizable lipids. Lipid 2 and lipid 6LNP showed dose-dependent expression of GFP protein (FIGS. 84A and 84B). However, at all doses tested, lipid 12 and lipid 13LNP performed poorly, probably because of non-optimal LNP surface charge properties and cytosol in T cells was reduced. In addition, lipid 2 and lipid 6LNP remained functional after being subjected to freeze-thaw stress, as illustrated in figure 85. As seen in fig. 83A and 83B, both compositions showed a slight change in particle size distribution after-80 ℃ frozen storage relative to particles stored at 4 ℃. In addition, both compositions retained the ability to bind and transfect primary human T cells after freeze thawing, with similar levels of% dii+ and DiI MFI values and similar levels of protein expression (% gfp+ cells and GFP MFI values) as observed after refrigerated (4 ℃) and frozen (-80 ℃) storage conditions.

Example 51-DiI T cell transfection experiments:

cd3+ T cells were isolated from frozen peripheral blood mononuclear cells using EasySep human T cell isolation kit on RoboSep automated cell isolation system from stem mel. T cells were plated into RPMI cell culture medium supplemented with glutamax, 10% fetal bovine serum, pen-strep, and 40ng/mL IL-2 in round bottom 96 well plates. Each well was seeded with 100 μl of cell suspension at a density of 1M T cells/mL (100K T cells/well). Cells were allowed to stand in an incubator at 37 ℃ for two hours and then transfected by gentle addition of 10 μl of 22 μg/mL (as mRNA) nanoparticle suspension, resulting in a final mRNA concentration of 2 μg/mL (unless otherwise indicated). The cells were gently mixed with a pipette and then incubated in an incubator at 37℃for 24 hours. After incubation, cells were diluted with FACS buffer (BD 554657) and analyzed using a BD Fortessa flow cytometer. The data was analyzed using FlowJo software from BD biosciences.

Examples 52-CD4 and CD69 staining

After 24 hours, cells were transferred to a 96-well conical bottom polypropylene plate and centrifuged at 350×g for 5 minutes. The supernatant was removed and transferred to a new conical bottom polypropylene plate for further analysis. The cells were washed by: 200. Mu.L of FACS buffer (BD 554657) was added, centrifuged at 350 Xg for 5min, and the supernatant was aspirated from each well. BV421 anti-human CD69 (BioLegend 310930 clone FN 50) and BV711 anti-human CD4 (BioLegend 344648 clone SK 3) antibodies were diluted 100-fold by adding 100. Mu.L of each antibody to 10mL of FACS buffer. mu.L of diluted antibody solution was added to each well and the plate was incubated for 20 minutes at room temperature. The plates were then washed by: centrifuge at 350 Xg for 5min, remove supernatant, re-suspend in 200. Mu.L FACS buffer, centrifuge at 350 Xg for 5min, and aspirate supernatant from each well. After washing, cells were resuspended in 100 μl of 1.6% formaldehyde and stored at 4 ℃ until FACS analysis. FACS analysis was performed using BD Fortessa equipped with a high-throughput sampler.

Example 53-human IFN-. Gamma.ELISA:

IFN-. Gamma.was measured using the R & D Duoset IL-2ELISA kit (PN DY 285B). Briefly, an Immulon 2hb 96 well plate (Thermo X1506319) was coated by: to each well was added 100. Mu.L of 2. Mu.g/mL of R & D IL-2 capture antibody solution, and the plate was incubated overnight at 4 ℃. Plates were washed three times with wash buffer (0.05 TWEEN-20, thermo 28360 in pH7.4TRIS buffered saline), blocked with reagent diluent (0.1% BSA in wash buffer) for one hour at room temperature, and then washed three more times with wash buffer. The supernatant was diluted three times in reagent diluent, and then 100 μl of the diluted supernatant was added to each well. IFN-gamma standards were prepared by serial dilution on the same plate. Plates were incubated for two hours at room temperature and washed three times with wash buffer. 100. Mu.L of detection antibody diluted in reagent diluent was added, incubated for 2 hours at room temperature, and the plate was then washed three times with wash buffer. 100. Mu.L of streptavidin-HRP was added and incubated for 20 min at room temperature, and the plates were washed three times with wash buffer. 100. Mu.L of substrate solution (Thermo N301) was added, incubated at room temperature for 20 minutes, and then the reaction was quenched by adding 100. Mu.L of stop solution (Invitrogen SS 04). The optical density at 450nm was read on a SpectraMax M5 plate reader. IFN-gamma concentrations were quantified relative to a standard curve based on IFN-gamma standards analyzed simultaneously.

Detailed description of the illustrated embodiments

1. A compound represented by formula I:

or a salt thereof, wherein:

n is 0 or 3;

o and p are independently integers selected from 2-6;

wherein the compound is not a compound selected from the group consisting of:

or a salt thereof.

2. A compound of embodiment 1 wherein o and p are 2.

3. A compound of embodiment 1 wherein o and p are 4.

4. A compound of embodiment 1 wherein o and p are 6.

5. The compound of any of embodiments 1-4, wherein X1 and X2 together form oxo, and X3 and X4 together form oxo.

6. The compound of any one of embodiments 1-4, wherein X1, X2, X3, and X4 are hydrogen.

7. The compound of any of embodiments 1-6, wherein Y is selected from-O-, -OC (O) -and-CH 2-.

8. The compound of embodiment 7 wherein Y is-O-.

9. A compound of embodiment 7 wherein Y is-OC (O) -.

10. The compound of embodiment 7 wherein Y is-CH 2-.

11. The compound of any one of embodiments 1-10, wherein R1 and R2 are independently C1-3 alkyl.

12. A compound of embodiment 11 wherein R1 and R2 are-CH 3.

13. A compound of embodiment 11 wherein R1 and R2 are-CH 2CH3.

14. The compound of any one of embodiments 1-13, wherein n is 0.

15. The compound of any one of embodiments 1-13, wherein n is 3.

16. A compound represented by formula II:

or a salt thereof, wherein:

n is 0 to 4;

wherein,

when both o and p are 1, r and s are independently 4, 5, 7 or 8, and

when both o and p are 2, r and s are independently 1, 2, 4 or 5.

17. The compound of embodiment 16 wherein X ¹ And X ² Together form oxo, and X ³ And X ⁴ Together forming oxo.

18. The compound of embodiment 16 or 17 wherein X ¹ 、X ² 、X ³ And X ⁴ Is hydrogen.

19. The compound of any of embodiments 16-18 wherein Y is selected from the group consisting of-O-, -OC (O) -and-CH ₂ -。

20. The compound of embodiment 19 wherein Y is-O-.

21. A compound of embodiment 19 wherein Y is-OC (O) -.

22. The compound of embodiment 19 wherein Y is-CH ₂ -。

23. The compound of any of embodiments 16-22, wherein R ¹ And R is ² Independently C _1-3 An alkyl group.

24. The compound of embodiment 23, wherein R ¹ And R is ² is-CH ₃ 。

25. The compound of embodiment 23, wherein R ¹ And R is ² is-CH ₂ CH ₃ 。

26. The compound of any one of embodiments 16-25, wherein n is 0.

27. The compound of any one of embodiments 16-25, wherein n is 3.

28. A compound selected from the group consisting of:

or a salt thereof.

29. The compound of embodiment 16, wherein the compound is of formula III:

or a salt thereof, wherein:

R ¹ and R is ² Independently C _1-3 Alkyl, or R ¹ And R is ² Forms an optionally substituted piperidinyl group together with the nitrogen atom;

X ¹ 、X ² 、X ³ And X ⁴ Is hydrogen or X ¹ And X ² Or X ³ And X ⁴ Together forming oxo; and is also provided with

n is an integer selected from 0-4.

30. The compound of embodiment 29 wherein R ¹ And R is ² Independently C _1-3 An alkyl group.

31. The compound of embodiment 29 or 30 wherein R ¹ And R is ² is-CH ₃ 。

32. The compound of any of embodiments 29-31, wherein Y is-O-.

33. The compound of any one of embodiments 29-32, wherein X ¹ And X ² Together form oxo, and X ³ And X ⁴ Together forming oxo.

34. The compound of any one of embodiments 29-33, wherein n is 3.

35. A compound of the formula:

or a salt thereof.

36. A Lipid Nanoparticle (LNP) comprising a lipid blend comprising a compound according to any one of embodiments 1-35 or a lipid of table 1.

37. The LNP of embodiment 36, wherein the lipid blend further comprises one or more of sterols, neutral phospholipids, PEG-lipids, and lipid-immune cell targeting group conjugates.

38. The LNP of embodiment 35 or 36, wherein the compound is present in the lipid blend in the range of 30-70-60 mole percent.

39. The LNP of any of embodiments 36-38, wherein the sterol (e.g., cholesterol) is present in the lipid blend in a range of 20-70 mole percent.

40. An LNP according to any of embodiments 36-39, wherein the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC).

41. An LNP according to any of embodiments 36-40, wherein the neutral phospholipid is present in the lipid blend in a range of 1-10 mole percent.

42. The LNP of any of embodiments 36-41, wherein the PEG-lipid is selected from the group consisting of PEG-distearoyl glycerol (PEG-DSG), PEG-dipalmitoyl glycerol (PEG-DAG, e.g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), and PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE).

43. The LNP of any one of embodiments 36-42, wherein the PEG-lipid is present in the lipid blend in a range of 1-10 mole percent.

44. The LNP of any one of embodiments 36-43, wherein the lipid-immune cell targeting group conjugate is present in the lipid blend in a range of 0.1-0.3 mole percent or 0.002-0.2 mole percent.

45. The LNP of embodiment 44, wherein the targeting group is a T cell targeting group.

46. The LNP of embodiment 45, wherein the T cell targeting moiety is an antibody or antigen binding fragment thereof that binds a T cell antigen.

47. The LNP of embodiment 46, wherein the T cell antigen is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, and T Cell Receptor (TCR) β (e.g., CD3 or CD 8).

48. The LNP of any one of embodiments 36-47, wherein the T cell targeting group is covalently coupled to the lipid via a linker comprising polyethylene glycol (PEG).

49. The LNP of embodiment 48, wherein the lipid is distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycerol-phosphoglyceride (DSPG), distearoyl-glycerol (DSG), dimyristoyl-glycerol (DMG), or ceramide.

50. The LNP of embodiment 48 or 49, wherein the PEG is selected from PEG 2000, PEG 1000, PEG 3000, PEG 3450, PEG 4000, or PEG 5000.

51. The LNP of any of embodiments 36-50, wherein the lipid blend further comprises free PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE), PEG-distearoyl-phosphatidylethanolamine (PEG-DMPE), N- (methylpolyoxycarbonyl) -1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1, 2-dimyristoyl-rac-glycerol-3-methylpolyethylene oxide (PEG-DMG), 1, 2-dipalmitoyl-rac-glycerol-3-methylpolyethylene oxide (PEG-dpp), 1, 2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG), 1, 2-distearoyl-rac-glycerol-3-methylpolyethylene oxide (PEG-DSPE), N-palmitoyl-sphingosine-1- { succinyl [ methoxy (polyethylene glycol) ] (PEG-ceramide), and DSPE-PEG-cysteine, or derivatives thereof.

52. The LNP of embodiment 51, wherein the derivative of PEG-lipid has a hydroxyl or carboxylic acid end group at the PEG terminus.

53. An LNP according to any of embodiments 36-52, wherein the LNP has an average diameter in the range of 50-200 nm.

54. The LNP of embodiment 53, wherein the LNP has an average diameter of about 100 nm.

55. An LNP according to any of embodiments 36-54, wherein the LNP has a polydispersity index in the range from 0.05 to 1.

56. An LNP according to any of embodiments 36-55, wherein the LNP has a zeta potential of from about-10 mV to about +30mV at pH 5.

57. An LNP according to any of embodiments 36-55, wherein the LNP has a zeta potential of from about-30 mV to about 5mV at pH 7.4.

58. An LNP according to any of embodiments 36-57, further comprising a nucleic acid disposed therein.

59. The LNP of embodiment 58, wherein the nucleic acid is DNA or RNA.

60. A Lipid Nanoparticle (LNP) comprising a lipid blend comprising a lipid-T cell targeting group conjugate and optionally a lipid as set forth in table 1.

61. The LNP of embodiment 60, wherein the T cell targeting moiety is an antibody that binds a T cell antigen.

62. The LNP of embodiment 61, wherein the T cell antigen is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, and T Cell Receptor (TCR) β.

63. The LNP of embodiment 62, wherein the T cell antigen is CD2, CD3, CD7 or CD8.

64. The LNP of any one of embodiments 60-63, wherein the T cell targeting group is covalently coupled to the lipid via a linker comprising polyethylene glycol (PEG).

65. The LNP of embodiment 64, wherein the lipid is distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycerol-phosphate glycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (dpp), or ceramide.

66. The LNP of embodiment 64 or 65, wherein the PEG is PEG 2000, PEG 1000, PEG 3000, PEG 3450, PEG 4000, or PEG 5000.

67. The LNP of any one of embodiments 60-66, wherein the lipid-T cell targeting group conjugate is present in the lipid blend in a range of 0.002-0.2 mole percent.

68. The LN of any one of embodiments 60-67 wherein the lipid blend further comprises one or more of cationic lipids, sterols, neutral phospholipids, and PEG-lipids.

69. The LNP of embodiment 68, wherein the ionizable cationic lipid is a compound according to any of embodiments 1-35 or a lipid set forth in table 1.

70. The LNP of embodiment 68 or 69, wherein the ionizable cationic lipid is present in the lipid blend in a range of 40-60 mole percent.

71. The LNP of any of embodiments 68-70, wherein the sterol (e.g., cholesterol) is present in the lipid blend in a range of 30-50 mole percent.

72. An LNP according to any of embodiments 68-71, wherein the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin.

73. An LNP according to any of embodiments 68-72, wherein the neutral phospholipid is present in the lipid blend in a range of 1-10 mole percent.

74. The LNP of any of embodiments 68-73, wherein the PEG-lipid is selected from PEG-distearoyl glycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e.g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE), and PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), N- (methylpolyoxycarbonyl) -1, 2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE-PEG), 1, 2-dipalmitoyl-rac-glycerol-3-methylpolyethylene oxide (PEG-DPPE), 1, 2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG), and N-palmitoyl-sphingosine-1- { succinyl [ methoxy (polyethylene glycol) ] (PEG-ceramide).

75. The LNP of any one of embodiments 68-74, wherein the PEG-lipid is present in the lipid blend in a range of 2-4 mole percent.

76. The LNP of any of embodiments 68-75, wherein the lipid blend further comprises free PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), N- (methylpolyoxycarbonyl) -1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1, 2-dimyristoyl-rac-glycerol-3-methylpolyethylene oxide (PEG-DMG), 1, 2-dipalmitoyl-rac-glycerol-3-methylpolyethylene oxide (PEG-DPG), 1, 2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG), 1, 2-distearoyl-rac-glycerol-3-methylpolyethylene oxide (PEG-DSPE), N-palmitoyl-sphingosine-1- { succinyl [ methoxy (polyethylene glycol) ] (PEG-ceramide), and DSPE-PEG-cysteine, or derivatives thereof.

77. An LNP according to any of embodiments 60-75, wherein the LNP has an average diameter in the range of 50-200 nm.

78. The LNP of embodiment 77, wherein the LNP has an average diameter of about 100 nm.

79. The LNP of any one of embodiments 60-78 wherein the LNP has a polydispersity index in the range from 0.05 to 1.

80. The LNP of any of embodiments 60-79, wherein the LNP has a zeta potential of from about-10 mV to about +30mV at pH 5.

81. The LNP of any of embodiments 60-80, further comprising a nucleic acid disposed therein.

82. The LNP of embodiment 81, wherein the nucleic acid is DNA or RNA (e.g., mRNA, tRNA, or siRNA).

83. The LNP of embodiment 81 or 82, wherein the number of nucleotides in the nucleic acid is from about 400 to about 6000.

84. A method of delivering a nucleic acid to an immune cell (e.g., a T cell), the method comprising exposing the immune cell to an LNP according to any one of embodiments 36-83 comprising the nucleic acid under conditions that allow the nucleic acid to enter the immune cell.

85. A method of delivering a nucleic acid to an immune cell (e.g., a T cell) of a subject in need thereof, the method comprising administering to the subject a composition comprising an LNP of any one of embodiments 36-83 comprising a nucleic acid, thereby delivering the nucleic acid to the immune cell.

86. A method of targeting delivery of a nucleic acid (e.g., mRNA) to an immune cell (e.g., T cell) of a subject, the method comprising administering to the subject an LNP according to any one of embodiments 36-83 containing the nucleic acid to facilitate targeted delivery of the nucleic acid to the immune cell.

Incorporated by reference

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described herein. All publications, scientific articles, patents, and patent publications cited are incorporated herein by reference in their entirety for all purposes.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the application is not entitled to antedate such publication by virtue of prior application.

Equivalent(s)

The present application may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are, therefore, to be considered in all respects illustrative rather than limiting on the application described herein. The scope of the application is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein. While the application has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Sequence listing

<110> Tide therapy Co

<120> ionizable cationic lipids and lipid nanoparticles and methods of synthesis and use thereof

<130> 18395-20340.40

<140> not yet allocated

<141> along with the submission

<150> US 63/172,024

<151> 2021-04-07

<150> US 63/169,395

<151> 2021-04-01

<150> US 63/169,296

<151> 2021-04-01

<150> US 63/166,205

<151> 2021-03-25

<150> US 63/121,801

<151> 2020-12-04

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr

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Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

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Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

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Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

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Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp

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Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

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Pro Lys Ser Ser Asp Lys Thr His Thr Cys

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Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly

20 25 30

Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn

85 90 95

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ser Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile Thr Asp Phe Tyr Pro

130 135 140

Gly Val Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro Val Thr Gln

145 150 155 160

Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn Lys Tyr Met

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Ala Ser Ser Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu Arg His Ser

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Ser Tyr Ser Cys Gln Val Thr His Glu Gly His Thr Val Glu Lys Ser

195 200 205

Leu Ser Arg Ala Asp Ser Ser

210 215

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Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly

20 25 30

Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn

85 90 95

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Val

145 150 155 160

Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

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Ser Tyr Ser Cys Arg Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Ala Glu Ser Ser

210 215

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp

210 215 220

Lys Thr His Thr Cys

225

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<213> artificial sequence

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Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Pro Gly Ala Lys Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser

35 40 45

Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

50 55 60

Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp

100 105 110

Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

115 120 125

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

130 135 140

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

145 150 155 160

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

165 170 175

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

180 185 190

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

195 200 205

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

210 215 220

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

225 230 235

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<213> artificial sequence

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Tyr Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Lys Pro His Tyr Asp Gly Tyr Tyr His Phe Phe Asp Ser Trp Gly

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Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

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Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

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Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

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Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser

210 215 220

Asp Lys Thr His Thr Cys

225 230

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Gly Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Thr Asp Ile Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Tyr Gln Tyr Asn Asn Gly Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Ser

210

<210> 8

<211> 227

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Asp Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr

210 215 220

His Thr Cys

225

<210> 9

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<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

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Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Arg Ser Ile Ser Gln Tyr

20 25 30

Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Asn Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 10

<211> 231

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

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Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Ser Asp Lys Thr His Thr Cys

225 230

<210> 11

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 11

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 12

<211> 227

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 12

Glu Ile Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Ser Phe

50 55 60

Lys Gly Arg Phe Thr Phe Ser Leu Asp Asp Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr

210 215 220

His Thr Cys

225

<210> 13

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr

65 70 75 80

Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 14

<211> 226

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Phe Thr Tyr Tyr Pro Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu

65 70 75 80

Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Asp Glu Val Arg Gly Tyr Leu Asp Val Trp Gly Ala Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys

225

<210> 15

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 15

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 16

<211> 226

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Met Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Gly Gly Gly Phe Thr Tyr Tyr Pro Asp Thr Val Lys

50 55 60

Gly Arg Phe Thr Leu Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Gln Gly Ala Asn Trp Glu Leu Val Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His

210 215 220

Thr Cys

225

<210> 17

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 17

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Phe Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Phe

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Phe Cys Gln Asn Gly His Asn Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 18

<211> 227

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 18

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Trp Ile His Trp Val Lys Gln Arg Pro Ile Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Gly Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Glu Asp Leu Tyr Tyr Ala Met Glu Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr

210 215 220

His Thr Cys

225

<210> 19

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 19

Asn Ile Met Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Val Tyr Tyr Cys His Gln

85 90 95

Tyr Leu Ser Ser His Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 20

<211> 229

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 20

Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Val Asn Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe

50 55 60

Thr Asp Lys Ala Ile Ser Thr Ile Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Thr Leu Thr Ser Asp Ala Ser Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Ser Pro Arg Asp Ser Ser Thr Asn Leu Ala Asp Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp

210 215 220

Lys Thr His Thr Cys

225

<210> 21

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 21

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 22

<211> 227

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Asp Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Arg Gly Ala Gly Ala Tyr Val Phe Asp His Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr

210 215 220

His Thr Cys

225

<210> 23

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 23

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Arg Ser Ile Ser Ala Ala

20 25 30

Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Asn Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 24

<211> 494

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 24

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Tyr Lys Ala Lys Glu Val Gln Leu Gln Gln Ser

20 25 30

Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys

35 40 45

Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln

50 55 60

Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn

65 70 75 80

Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr

85 90 95

Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

100 105 110

Ser Glu Asp Ser Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly

115 120 125

Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr

130 135 140

Val Ser Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro

165 170 175

Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr

180 185 190

Met Asp Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile

195 200 205

Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly

210 215 220

Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala

225 230 235 240

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro

245 250 255

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

260 265 270

Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

275 280 285

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

290 295 300

Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

340 345 350

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

355 360 365

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val

370 375 380

Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly Gln Asn

385 390 395 400

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

405 410 415

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

420 425 430

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

435 440 445

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

450 455 460

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

465 470 475 480

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 25

<211> 1491

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 25

atggagaccg acaccctgtt gctttgggta ctgttacttt gggtgcccgg atctaccggt 60

gattacaagg ccaaggaggt gcagctgcag cagagcggag ccgagctggt gaagccaggc 120

gcttccgtga agatgtcttg taaggcctcc ggctacacat tcaccagcta caatatgcac 180

tgggtaaagc agactccggg gcagggcctg gagtggatag gtgccatcta ccctggcaac 240

ggcgacacca gctacaacca gaagtttaag gggaaggcta ctctaacagc ggacaagtcg 300

tcctctaccg cctacatgca actcagctcc ctgacgagcg aggactccgc ggactactac 360

tgtgcccgct ccaactacta cggctctagc tattggttct tcgacgtgtg gggcgctgga 420

acgaccgtga ccgtgtcttc cggtggaggt tccgggggcg gaagcggcgg tggcggcagt 480

tcggacatcg tgctgaccca gagccctgcc atcctgtccg cttccccggg ggagaaagtt 540

acgatgacct gccgagcgag ctccagtgtc aactacatgg attggtacca gaagaagccc 600

ggcagcagtc ccaagccgtg gatttacgct actagcaacc tggcgtccgg tgtcccggct 660

cgcttctcag gttctggctc gggtactagt tattcattaa ccatttctcg cgtggaggct 720

gaggacgctg ccacctacta ctgccaacag tggtctttca accctcccac tttcggaggc 780

ggcaccaagc tcgagatcaa gggcgggggt ggctccgcag cagccattga ggtgatgtat 840

cctcctccct atttggacaa cgagaagtca aatggcacca tcatccacgt taagggcaag 900

cacctgtgcc catctcccct gttcccaggc ccctctaagc ccttctgggt cctggtggtg 960

gtcggcggcg tcctggcatg ttactctctg ctggtgaccg tcgcgttcat catcttttgg 1020

gtccggtcca agcgcagccg cctgctccac tccgactaca tgaatatgac tcctcgtagg 1080

cccggtccaa cccgcaagca ctaccagccg tacgcgccgc ccagagactt tgctgcttac 1140

cgatccagag tgaaattttc taggtcggcc gaacctcccg catatcagca gggccagaac 1200

cagctgtaca acgaactcaa cttgggacgg cgcgaggaat acgatgtgct ggataaacgc 1260

cgtggccgcg atcccgagat gggcgggaag ccacgtcgca aaaaccctca ggagggcctt 1320

tacaacgagt tgcagaagga caaaatggcg gaggcctact ccgagatcgg aatgaagggg 1380

gagcgccggc gcggcaaagg gcatgacggc ctctaccagg gcctgtccac agccacgaaa 1440

gacacctatg acgccctgca tatgcaggcc ctgcccccgc gctgataatg a 1491

<210> 26

<211> 248

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 26

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Asn Trp Ile Arg

35 40 45

Gln Ser Pro Ser Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser

50 55 60

Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile

65 70 75 80

Asn Pro Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val

85 90 95

Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Asn Ile Ala

100 105 110

Ala Trp Thr Trp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr

115 120 125

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

130 135 140

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

145 150 155 160

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

165 170 175

Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu Gln Ser Ser Gly

180 185 190

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

195 200 205

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

210 215 220

Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys

225 230 235 240

Gly Gly His His His His His His

245

<210> 27

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 27

Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Thr Ser Leu Arg Ala Thr Asp Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 28

<211> 238

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 28

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Ser Ser Gly Tyr Ser Gly Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser

210 215 220

Asp Lys Thr His Thr Cys Gly Gly His His His His His His

225 230 235

<210> 29

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 29

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Gly Glu Asp Val Gly Asp Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Ser Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 30

<211> 238

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 30

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Ser Ser Gly Tyr Ser Gly Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser

210 215 220

Asp Lys Thr His Thr Cys Gly Gly His His His His His His

225 230 235

<210> 31

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 31

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Phe Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Gln Thr Pro Trp Thr Phe Gly His Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 32

<211> 233

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 32

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Ser Phe Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Arg Lys Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr

210 215 220

His Thr Cys His His His His His His

225 230

<210> 33

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 33

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile Ile His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro His Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 34

<211> 232

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 34

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Gly Gly Phe Leu Tyr Tyr Leu Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu

65 70 75 80

His Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Ser Ser Tyr Gly Glu Ile Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His

210 215 220

Thr Cys His His His His His His

225 230

<210> 35

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 35

Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Arg Ile Gly Thr Ser

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Thr Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Val Ala Asp Tyr Tyr Cys Gln Gln Ser His Gly Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 36

<211> 233

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 36

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Val Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr

210 215 220

His Thr Cys His His His His His His

225 230

<210> 37

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 37

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Gln Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asp Gly His Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 38

<211> 234

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Ser Asp Lys Thr His Thr Cys

225 230

<210> 39

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 39

Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly

20 25 30

Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn

85 90 95

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Val

145 150 155 160

Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Arg Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Ala Glu Ser Ser

210 215

<210> 40

<211> 234

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 40

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys

225 230

<210> 41

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 41

Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly

20 25 30

Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn

85 90 95

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Val

145 150 155 160

Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Arg Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Ala Glu Cys Ser

210 215

<210> 42

<211> 226

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Met Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Gly Gly Gly Phe Thr Tyr Tyr Pro Asp Thr Val Lys

50 55 60

Gly Arg Phe Thr Leu Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Gln Gly Ala Asn Trp Glu Leu Val Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys

225

<210> 43

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 43

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Phe Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Phe

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Phe Cys Gln Asn Gly His Asn Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 44

<211> 227

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 44

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Trp Ile His Trp Val Lys Gln Arg Pro Ile Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Gly Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Glu Asp Leu Tyr Tyr Ala Met Glu Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys

225

<210> 45

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 45

Asn Ile Met Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Val Tyr Tyr Cys His Gln

85 90 95

Tyr Leu Ser Ser His Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 46

<211> 240

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Cys Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Ser Asp Lys Thr His Thr Cys His His His His His His

225 230 235 240

<210> 47

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 47

Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly

20 25 30

Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn

85 90 95

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Val

145 150 155 160

Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Cys Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Arg Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Ala Glu Ser Ser

210 215

<210> 48

<211> 234

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 48

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp Asn Tyr Ser Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys

210 215 220

Thr His Thr Cys His His His His His His

225 230

<210> 49

<211> 213

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 49

Gln Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Ser

210

<210> 50

<211> 234

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 50

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Pro Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Thr Ser Gly Gly Arg Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Phe Arg Gln Tyr Ser Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys

210 215 220

Thr His Thr Cys His His His His His His

225 230

<210> 51

<211> 216

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 51

Asp Ile Gln Leu Thr Gln Pro Asn Ser Val Ser Thr Ser Leu Gly Ser

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Leu Ser Ser Gly Asn Ile Glu Asn Asn

20 25 30

Tyr Val His Trp Tyr Gln Leu Tyr Glu Gly Arg Ser Pro Thr Thr Met

35 40 45

Ile Tyr Asp Asp Asp Lys Arg Pro Asp Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala Phe Leu Thr Ile His Asn

65 70 75 80

Val Ala Ile Glu Asp Glu Ala Ile Tyr Phe Cys His Ser Tyr Val Ser

85 90 95

Ser Phe Asn Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Cys Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Ser Ser

210 215

<210> 52

<211> 237

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 52

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Asn Pro Thr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Ser Asp Lys Thr His Thr Cys His His His His His His

225 230 235

<210> 53

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 53

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 54

<211> 234

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 54

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Pro Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys

210 215 220

Thr His Thr Cys His His His His His His

225 230

<210> 55

<211> 213

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 55

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Ser

210

<210> 56

<211> 230

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 56

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Tyr Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Pro His Tyr Asp Gly Tyr Tyr His Phe Phe Asp Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser

210 215 220

Asp Lys Thr His Thr Cys

225 230

<210> 57

<211> 213

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 57

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Gly Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Thr Asp Ile Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Tyr Gln Tyr Asn Asn Gly Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Ser

210

<210> 58

<211> 226

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 58

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Ala

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Thr Pro Gly Lys Pro Met Glu Trp Ile

35 40 45

Gly His Ile Lys Tyr Asp Gly Ser Tyr Thr Asn Tyr Ala Pro Ser Leu

50 55 60

Lys Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Asn Val Arg Ser Glu Asp Ser Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Pro Gly Ala Ala Ser Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His

210 215 220

Thr Cys

225

<210> 59

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 59

Asp Val Val Leu Thr Gln Thr Pro Val Ala Gln Pro Val Thr Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ile Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Pro Glu Asp Trp Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Thr His Asp Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 60

<211> 229

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 60

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Arg Thr Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Lys Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe

50 55 60

Gln Asp Lys Ala Thr Val Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Thr Tyr Ala Tyr Asp Gly Asn Trp Tyr Phe Asp Val Trp Gly Ala

100 105 110

Gly Thr Ala Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp

210 215 220

Lys Thr His Thr Cys

225

<210> 61

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 61

Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Val Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Phe

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr

65 70 75 80

Glu Asp Met Glu Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 62

<211> 230

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 62

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Ile Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Lys Ala Ile Leu Ser Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Ile Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Arg Asp Ala Lys Tyr Asp Gly Tyr Ala Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser

210 215 220

Asp Lys Thr His Thr Cys

225 230

<210> 63

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 63

Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly

1 5 10 15

Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Thr Leu Leu His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Val Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asn Arg Phe Ser Gly Ser Gly Ser Glu Thr Thr Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Glu

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 64

<211> 236

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 64

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly His

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ile Tyr Phe Tyr Gly Thr Thr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser

210 215 220

Asp Lys Thr His Thr Cys His His His His His His

225 230 235

<210> 65

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 65

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Thr Ile Ser Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 66

<211> 235

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 66

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ala Ile Asp Asn Asp Gly Gly Ser Ile Ser Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Gln Gly Arg Leu Arg Arg Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Thr Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp

210 215 220

Lys Thr His Thr Cys His His His His His His

225 230 235

<210> 67

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 67

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 68

<211> 239

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 68

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Met His Trp Met Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Asn Pro Lys Asn Gly Gly Thr Ser His Asn Gln Arg Phe

50 55 60

Met Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Arg Gly Leu Asn Tyr Gly Phe Asp Val Arg Tyr Phe Asp

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Cys Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Ser Asp Lys Thr His Thr Cys His His His His His His

225 230 235

<210> 69

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 69

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 70

<211> 237

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 70

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Ser Asp Lys Thr His Thr Cys His His His His His His

225 230 235

<210> 71

<211> 481

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 71

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser Gly Gly Gly Gly Ser

210 215 220

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu

225 230 235 240

Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys

245 250 255

Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe Gly Met Asn Trp Val Arg

260 265 270

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Tyr Tyr Asp

275 280 285

Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

290 295 300

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

305 310 315 320

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Pro His Tyr Asp

325 330 335

Gly Tyr Tyr His Phe Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr

340 345 350

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

355 360 365

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

370 375 380

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Gly

385 390 395 400

Ser Gln Asp Ile Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly

405 410 415

Lys Ala Pro Lys Leu Leu Ile Tyr Asn Thr Asp Ile Leu His Thr Gly

420 425 430

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe

435 440 445

Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Tyr

450 455 460

Gln Tyr Asn Asn Gly Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

465 470 475 480

Lys

<210> 72

<211> 231

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 72

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Ser Asp Lys Thr His Thr Cys

225 230

<210> 73

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 73

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 74

<211> 235

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 74

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Asp His Ser Thr Asn Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Lys Tyr Gly Gly Asp Tyr Asp Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp

210 215 220

Lys Thr His Thr Cys His His His His His His

225 230 235

<210> 75

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 75

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Ser

210

<210> 76

<211> 136

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 76

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Gly Thr Ser Gly Arg Thr Phe Asn Val Met

20 25 30

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala

35 40 45

Val Arg Trp Ser Ser Thr Gly Ile Tyr Tyr Thr Gln Tyr Ala Asp Ser

50 55 60

Val Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Tyr Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Asp Thr Tyr Asn Ser Asn Pro Ala Arg Trp Asp Gly Tyr

100 105 110

Asp Phe Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Cys

115 120 125

Gly Gly His His His His His His

130 135

<210> 77

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 77

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Asp Tyr

20 25 30

Gly Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val

35 40 45

Ala Asp Ile Asp Trp Asn Gly Glu His Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ala Thr Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Ala Leu Pro Tyr Thr Val Arg Lys Tyr Asn Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Cys Gly Gly His His

115 120 125

His His His His

130

<210> 78

<211> 138

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 78

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Pro Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Tyr Arg Gly Tyr

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Val Trp Ser Gly Gly Asn Thr Tyr Tyr Glu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Met Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Ser Ala Thr Tyr Tyr Cys

85 90 95

Ala Ala Lys Ile Arg Pro Tyr Ile Phe Lys Ile Ala Gly Gln Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gly Gly Gly

115 120 125

Ser Gly Gly His His His His His His Cys

130 135

<210> 79

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 79

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Ile Tyr Lys Ser Phe

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val

35 40 45

Ala Val Ile Gly Ser Arg Gly Asn Asn Arg Gly Arg Thr Asn Tyr Ala

50 55 60

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Thr Gly Asn

65 70 75 80

Thr Val Tyr Leu Leu Met Asn Lys Leu Arg Pro Glu Asp Thr Ala Ile

85 90 95

Tyr Tyr Cys Asn Thr Ala Pro Leu Val Ala Gly Arg Pro Trp Gly Arg

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ser Gly Gly His His

115 120 125

His His His His Cys

130

<210> 80

<211> 138

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 80

Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Pro Tyr Ser Ser Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Ile Asp Ser Asp Gly Arg Thr Arg Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Arg Met Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Arg Phe Gly Pro Met Gly Cys Val Asp Leu Ser Thr Leu Ser Phe

100 105 110

Gly His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ile Thr Gly Gly

115 120 125

Gly Cys His His His His His His His His

130 135

<210> 81

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 81

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Val His Lys Ile Asn

20 25 30

Phe Tyr Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Ala Tyr Tyr Cys Arg

85 90 95

Ala Leu Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Cys Gly Gly His His His His His His

115 120 125

<210> 82

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 82

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Arg Leu His

20 25 30

Thr Met Glu Trp Tyr Arg Arg Thr Pro Glu Thr Gln Arg Glu Trp Val

35 40 45

Ala Thr Ile Thr Ser Gly Gly Thr Thr Asn Tyr Pro Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asp Thr Lys Lys Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His

85 90 95

Ala Val Ala Thr Glu Asp Ala Gly Phe Pro Pro Ser Asn Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Cys Gly Gly His His

115 120 125

His His His His

130

<210> 83

<211> 135

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 83

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Pro Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Tyr Arg Gly Tyr

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Val Trp Ser Asp Gly Asn Thr Tyr Tyr Glu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Ser Ala Thr Tyr Tyr Cys

85 90 95

Ala Ala Lys Ile Arg Pro Tyr Ile Phe Lys Ile Ala Gly Gln Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Cys Gly

115 120 125

Gly His His His His His His

130 135

<210> 84

<211> 235

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 84

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Tyr Ala Tyr

20 25 30

Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Lys Lys Asp Gly Thr Thr Ile Asn Tyr Thr Pro Ser Leu

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Lys Leu Gly Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Glu Glu Arg Asp Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Val

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr

210 215 220

His Thr Cys Gly Gly His His His His His His

225 230 235

<210> 85

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 85

Gln Phe Val Leu Thr Gln Pro Asn Ser Val Ser Thr Asn Leu Gly Ser

1 5 10 15

Thr Val Lys Leu Ser Cys Lys Arg Ser Thr Gly Asn Ile Gly Ser Asn

20 25 30

Tyr Val Asn Trp Tyr Gln Gln His Glu Gly Arg Ser Pro Thr Thr Met

35 40 45

Ile Tyr Arg Asp Asp Lys Arg Pro Asp Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala Leu Leu Thr Ile Asn Asn

65 70 75 80

Val Gln Thr Glu Asp Glu Ala Asp Tyr Phe Cys Gln Ser Tyr Ser Ser

85 90 95

Gly Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Val

145 150 155 160

Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Cys Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Arg Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Ala Glu Ser Ser

210 215

<210> 86

<211> 229

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 86

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Ile Gln Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Asn Pro Tyr Asn Asp Tyr Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ile Thr Ala Tyr

65 70 75 80

Met Glu Phe Ser Leu Thr Ser Glu Asp Ser Ala Leu Tyr Cys Ala Arg

85 90 95

Trp Gly Asp Gly Asn Tyr Trp Gly Arg Gly Thr Leu Thr Val Ser Ser

100 105 110

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

115 120 125

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

130 135 140

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

145 150 155 160

Gly Val His Thr Cys Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

165 170 175

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

180 185 190

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

195 200 205

Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Gly Gly His

210 215 220

His His His His His

225

<210> 87

<211> 211

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 87

Asp Ile Glu Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Tyr Phe His Trp Tyr Gln Lys Pro Gly Ser Ser Pro Lys Leu Cys Ile

35 40 45

Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Ser Thr Ser Tyr Ser Leu Thr Ile Ser Met Glu Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Phe Cys His Gln Tyr His Arg Ser Pro Thr Phe Gly

85 90 95

Gly Gly Thr Lys Leu Glu Thr Lys Arg Thr Val Ala Ala Pro Ser Val

100 105 110

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

115 120 125

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

130 135 140

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

145 150 155 160

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys Ser Thr Leu

165 170 175

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

180 185 190

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

195 200 205

Gly Glu Ser

210

<210> 88

<211> 238

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 88

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Gly Asp Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Tyr Ser Asn Phe His Tyr Tyr Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser

210 215 220

Asp Lys Thr His Thr Cys Gly Gly His His His His His His

225 230 235

<210> 89

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 89

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Ser Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 90

<211> 237

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 90

Gln Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp

210 215 220

Lys Thr His Thr Cys Gly Gly His His His His His His

225 230 235

<210> 91

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 91

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Ser Leu Asn Ile His

65 70 75 80

Pro Val Asp Glu Asp Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 92

<211> 234

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 92

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu Trp Leu Gly

35 40 45

Val Ile Trp Pro Gly Thr Asn Phe Asn Ser Ala Leu Met Ser Arg Leu

50 55 60

Thr Ile Ser Glu Asp Thr Ser Lys Asn Gln Val Ser Leu Lys Leu Ser

65 70 75 80

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Cys Ala Arg Asp Arg Ala

85 90 95

Tyr Gly Asn Tyr Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His

210 215 220

Thr Cys Gly Gly His His His His His His

225 230

<210> 93

<211> 210

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 93

Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Ser Ala Ser Val Gly Asp

1 5 10 15

Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr Val Thr

20 25 30

Ser Leu Met Gln Trp Tyr Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ala Ala Ser Asn Val Asp Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Gln Pro Glu Asp

65 70 75 80

Ile Ala Thr Tyr Cys Gln Ser Arg Lys Val Pro Phe Thr Phe Gly Gly

85 90 95

Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Ser

210

<210> 94

<211> 233

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 94

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Lys Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Tyr Ser Gly Ser Ser Asp Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Ala Thr Leu Thr Val Asp Asn Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr

210 215 220

Cys Gly Gly His His His His His His

225 230

<210> 95

<211> 213

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 95

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala

20 25 30

Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Asn Ile Leu Gly Thr Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Cys Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Ser

210

<210> 96

<211> 234

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 96

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ile Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val

35 40 45

Ala Ala Ile Asn Pro Asp Gly Gly Asn Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Gly Pro Gly Phe Asp Ser Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Cys Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His

210 215 220

Thr Cys Gly Gly His His His His His His

225 230

<210> 97

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 97

Glu Asn Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Arg Asn Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Phe Pro Gly Ser Gly Phe Pro Phe Met

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Cys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Ser

210 215

<210> 98

<211> 262

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 98

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Tyr Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Pro His Tyr Asp Gly Tyr Tyr His Phe Phe Asp Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Lys Gly Ser Gln Asp Ile Asn Asn Tyr Leu Ala Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asn Thr

180 185 190

Asp Ile Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile

210 215 220

Ala Thr Tyr Tyr Cys Tyr Gln Tyr Asn Asn Gly Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Ser Gly Gly Cys Gly Gly

245 250 255

His His His His His His

260

<210> 99

<211> 243

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 99

Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Pro Tyr Ser Ser Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Ile Asp Ser Asp Gly Arg Thr Arg Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Arg Met Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Arg Phe Gly Pro Met Gly Cys Val Asp Leu Ser Thr Leu Ser Phe

100 105 110

Gly His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ile Thr Ala Ser

115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

145 150 155 160

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

165 170 175

His Thr Cys Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

180 185 190

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

195 200 205

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

210 215 220

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Gly Gly His His His

225 230 235 240

His His His

<210> 100

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 100

Gly Ser Thr Phe Ser Asp Tyr Gly

1 5

<210> 101

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 101

Ile Asp Trp Asn Gly Glu His Thr

1 5

<210> 102

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 102

Ala Ala Asp Ala Leu Pro Tyr Thr Val Arg Lys Tyr Asn Tyr

1 5 10

<210> 103

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 103

Lys Glu Arg Glu

1

<210> 104

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 104

Lys Gln Arg Glu

1

<210> 105

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 105

Gly Leu Glu Trp

1

<210> 106

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 106

Lys Glu Arg Glu Leu

1 5

<210> 107

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 107

Lys Glu Arg Glu Phe

1 5

<210> 108

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 108

Lys Gln Arg Glu Leu

1 5

<210> 109

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 109

Lys Gln Arg Glu Phe

1 5

<210> 110

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 110

Lys Glu Arg Glu Gly

1 5

<210> 111

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 111

Lys Gln Arg Glu Trp

1 5

<210> 112

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 112

Lys Gln Arg Glu Gly

1 5

<210> 113

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 113

Thr Glu Arg Glu

1

<210> 114

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 114

Thr Glu Arg Glu Leu

1 5

<210> 115

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 115

Thr Gln Arg Glu

1

<210> 116

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 116

Thr Gln Arg Glu Leu

1 5

<210> 117

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 117

Lys Glu Cys Glu

1

<210> 118

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 118

Lys Glu Cys Glu Leu

1 5

<210> 119

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 119

Lys Glu Cys Glu Arg

1 5

<210> 120

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 120

Lys Gln Cys Glu

1

<210> 121

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 121

Lys Gln Cys Glu Leu

1 5

<210> 122

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 122

Arg Glu Arg Glu

1

<210> 123

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 123

Arg Glu Arg Glu Gly

1 5

<210> 124

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 124

Arg Gln Arg Glu

1

<210> 125

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 125

Arg Gln Arg Glu Leu

1 5

<210> 126

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 126

Arg Gln Arg Glu Phe

1 5

<210> 127

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 127

Arg Gln Arg Glu Trp

1 5

<210> 128

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 128

Gln Glu Arg Glu

1

<210> 129

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 129

Gln Glu Arg Glu Gly

1 5

<210> 130

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 130

Gln Gln Arg Glu

1

<210> 131

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 131

Gln Gln Arg Glu Trp

1 5

<210> 132

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 132

Gln Gln Arg Glu Leu

1 5

<210> 133

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 133

Gln Gln Arg Glu Phe

1 5

<210> 134

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 134

Lys Gly Arg Glu

1

<210> 135

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 135

Lys Gly Arg Glu Gly

1 5

<210> 136

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 136

Lys Asp Arg Glu

1

<210> 137

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 137

Lys Asp Arg Glu Val

1 5

<210> 138

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 138

Asp Glu Cys Lys Leu

1 5

<210> 139

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 139

Asn Val Cys Glu Leu

1 5

<210> 140

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 140

Gly Val Glu Trp

1

<210> 141

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 141

Glu Pro Glu Trp

1

<210> 142

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 142

Gly Leu Glu Arg

1

<210> 143

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 143

Asp Gln Glu Trp

1

<210> 144

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 144

Asp Leu Glu Trp

1

<210> 145

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 145

Gly Ile Glu Trp

1

<210> 146

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 146

Glu Leu Glu Trp

1

<210> 147

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 147

Gly Pro Glu Trp

1

<210> 148

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 148

Glu Trp Leu Pro

1

<210> 149

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 149

Gly Pro Glu Arg

1

<210> 150

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<220>

<221> variant

<222> 1, 2, 3, 4, 5, 6, 7, 8, 9

<223> may or may not be present

<220>

<221> variant

<222> 1, 2, 3, 4, 5, 6, 7, 8, 9, 10

<223> Xaa = a preferred naturally occurring amino acid residue independently selected from

<400> 150

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10

<210> 151

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<220>

<221> variant

<222> 1, 2, 3, 4

<223> may or may not be present

<220>

<221> variant

<222> 1, 2, 3, 4, 5

<223> Xaa = naturally occurring amino acid, preferably not cysteine

<400> 151

Xaa Xaa Xaa Xaa Xaa

1 5

<210> 152

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<220>

<221> variant

<222> (1)..(5)

<223> may repeat 1, 2, 3, 4, 5, 6, 7 or more occurrences

<400> 152

Gly Gly Gly Gly Ser

1 5

<210> 153

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 153

Ala Ala Ala

1

<210> 154

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 154

Gly Gly Gly Gly Ser

1 5

<210> 155

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 155

Ser Gly Gly Ser Gly Gly Ser

1 5

<210> 156

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 156

Gly Gly Gly Gly Ser Gly Gly Ser

1 5

<210> 157

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 157

Gly Gly Gly Gly Ser Gly Gly Gly Ser

1 5

<210> 158

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 158

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 159

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 159

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 160

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Ser

<210> 161

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 161

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 162

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 162

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 163

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 163

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 164

<211> 35

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 164

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser

35

<210> 165

<211> 40

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 165

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser

35 40

<210> 166

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 166

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 167

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 167

Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys

1 5 10 15

Thr His Thr Cys Pro Pro Cys Pro

20

<210> 168

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 168

Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala

1 5 10

<210> 169

<211> 62

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 169

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu

35 40 45

Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

50 55 60

Claims

1. A Lipid Nanoparticle (LNP) for targeted delivery of a nucleic acid to an immune cell comprising a lipid blend comprising:

(a) A lipid-immune cell targeting group conjugate comprising a compound of formula IV: [ lipid ] - [ optional linker ] - [ immune cell targeting group ], and

(b) An ionizable cationic lipid comprising

Wherein the LNP further comprises a nucleic acid disposed therein.

2. The LNP of claim 1, wherein the immune cell targeting group comprises an antibody that binds a T cell antigen.

3. The LNP of claim 2, wherein the T cell antigen is CD3, CD4, CD7, CD8, or a combination thereof (e.g., both CD3 and CD8, both CD4 and CD8, or both CD7 and CD 8).

4. The LNP of claim 2, wherein the immune cell targeting group comprises an antibody that binds a Natural Killer (NK) cell antigen.

5. The LNP of claim 4, wherein the NK cell antigen is CD7, CD8, CD56, or a combination thereof (e.g., both CD7 and CD 8).

6. The LNP of any one of claims 1 to 5, wherein the immune cell targeting group is covalently coupled to a lipid in the lipid blend via a linker comprising polyethylene glycol (PEG).

7. The LNP of claim 6, wherein the lipid covalently coupled to the immune cell targeting group via a PEG-containing linker is distearoyl glycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycerol-phosphate glycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (dpp), or ceramide.

8. The LNP of claim 6 or 7, wherein said PEG is PEG 2000 or PEG 3400.

9. The LNP of any one of claims 1-8, wherein the lipid-immune cell targeting group conjugate is present in the lipid blend in a range of 0.001-0.5 mole percent (e.g., 0.002-0.2 mole percent).

10. The LNP of any one of claims 1-9, wherein the lipid blend further comprises one or more of a structural lipid (e.g., a sterol), a neutral phospholipid, and a free PEG-lipid.

11. The LNP of any one of claims 1-10, wherein the ionizable cationic lipid is present in the lipid blend in a range of 30-70 (e.g., 40-60) mole percent.

12. The LNP of claim 10, wherein the sterol is present in the lipid blend in a range of 20-70 (e.g., 30-50) mole percent.

13. LNP according to claim 10 or 12, wherein the sterol is cholesterol.

14. LNP according to any one of claims 10 to 13, wherein the neutral phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), sphingomyelin.

15. LNP according to any one of claims 10 to 14, wherein the neutral phospholipids are present in the lipid blend in the range of 1-10 mole percent.

16. The LNP of any one of claims 10 to 15, wherein the free PEG-lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, the PEG lipid may be PEG-dioleoyl glycerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-dppg), PEG-diiodoyl-glycerol-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoyl glycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycerol-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycerol-phosphoethanolamine (PEG-DOPE), 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide, or PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

17. The LNP of any one of claims 10-15, wherein the free PEG-lipid comprises a diacyl phosphatidylethanolamine comprising a dipalmitoyl (C16) chain or a distearoyl (C18) chain.

18. The LNP of any one of claims 10-17, wherein the free PEG-lipid is present in the lipid blend in a range of 1-4 mole percent.

19. The LNP of any one of claims 10 to 18, wherein the free PEG-lipid comprises the same or different lipid as the lipid in the lipid-immune cell targeting group conjugate.

20. An LNP according to any one of claims 1 to 19, wherein the LNP has an average diameter in the range of 50-200 nm.

21. The LNP of claim 20 wherein the LNP has an average diameter of about 100 nm.

22. The LNP of any one of claims 1-21 wherein the LNP has a polydispersity index in a range from 0.05 to 1.

23. The LNP of any one of claims 1-22 wherein the LNP has a zeta potential of from about-10 mV to about +30mV at pH 5.

24. The LNP of any one of claims 1-23, wherein the nucleic acid is DNA or RNA.

25. The LNP of claim 24, wherein said RNA is mRNA.

26. The LNP of claim 25, wherein the mRNA encodes a receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen, an enzyme, or a vaccine.

27. The LNP of claim 25, wherein said mRNA encodes a polypeptide capable of modulating an immune response in said immune cell.

28. The LNP of claim 25, wherein said mRNA encodes a polypeptide capable of reprogramming said immune cell.

29. The LNP of claim 27, wherein the mRNA encodes a synthetic T cell receptor (synTCR) or a Chimeric Antigen Receptor (CAR).

30. The LNP of any one of claims 1-29, wherein the immune cell targeting group comprises an antibody, and the antibody is a Fab or immunoglobulin single variable domain (e.g., nanobody).

31. The LNP of any one of claims 1-29, wherein the immune cell targeting group comprises a Fab, F (ab ') 2, fab' -SH, fv, or scFv fragment.

32. The LNP of claim 30 or claim 31, wherein the immune cell targeting group comprises a Fab engineered to knock out the natural interchain disulfide bond at the C-terminus.

33. The LNP of claim 32, wherein said Fab comprises a heavy chain fragment comprising a C233S substitution and a light chain fragment comprising a C214S substitution, numbered according to Kabat.

34. The LNP of any one of claims 31-33, wherein the immune cell targeting group comprises a Fab having non-native interchain disulfide bonds (e.g., engineered embedded interchain disulfide bonds).

35. The LNP of claim 34, wherein the Fab comprises an F174C substitution in the heavy chain fragment and an S176C substitution in the light chain fragment, numbered according to Kabat.

36. The LNP of claims 31-35, wherein the immune cell targeting group comprises a Fab containing a cysteine at the C-terminus of the heavy or light chain fragment.

37. The LNP of claim 36, wherein said Fab further comprises one or more amino acids between the heavy chain fragment of said Fab and said C-terminal cysteine.

38. The LNP of claim 30, wherein the immune cell targeting group comprises an immunoglobulin single variable domain.

39. An LNP according to claim 30 or claim 38, wherein the immunoglobulin single variable domain comprises a cysteine at the C-terminus.

40. The LNP of claim 39, wherein the immunoglobulin single variable domain comprises a VHH domain, and further comprises a spacer comprising one or more amino acids between the VHH domain and the C-terminal cysteine.

41. The LNP of any one of claims 31 and 38-40, wherein the immune cell targeting group comprises two or more VHH domains.

42. The LNP of claim 41, wherein the two or more V' s _HH The domains are linked by amino acid linkers.

43. The LNP of claim 41, wherein the immune cell targeting group comprises a first V linked to an antibody CH1 domain _HH Domain and constant domain with antibody light chainSecond V of connection _HH A domain, and wherein the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds.

44. The LNP of any one of claims 30 and 38-40, wherein the immune cell targeting group comprises a VHH domain linked to an antibody CH1 domain, and wherein the antibody CH1 domain is linked to an antibody light chain constant domain by one or more disulfide bonds.

45. The LNP of claim 43 or 44, wherein the CH1 domain comprises F174C and C233S substitutions and the light chain constant domain comprises S176C and C214S substitutions, numbered according to Kabat.

46. The LNP of any one of claims 1-27, wherein the immune cell targeting group comprises a Fab comprising:

(a) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO. 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO. 2 or 3; or alternatively

47. A method of targeting delivery of a nucleic acid to an immune cell of a subject, the method comprising contacting the immune cell with a Lipid Nanoparticle (LNP), wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising a compound of the formula:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the LNP provides at least one of the following benefits:

(iii) Increased transfection efficiency compared to reference LNP; and

48. A method of expressing a polypeptide of interest in a targeted immune cell of a subject, the method comprising contacting the immune cell with a Lipid Nanoparticle (LNP), wherein the LNP comprises:

(a) An ionizable cationic lipid;

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids

(e) Free polyethylene glycol (PEG) lipids, and

(f) Nucleic acids encoding the polypeptides.

49. A method as defined in claim 48 wherein the LNP provides at least one of the following benefits:

(iv) Increased transfection efficiency compared to reference LNP; and

50. A method of modulating cellular function of a target immune cell in a subject, the method comprising administering to the subject a Lipid Nanoparticle (LNP), wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) Nucleic acids encoding polypeptides for modulating cellular functions of the immune cells.

51. The method of claim 50 wherein the LNP provides at least one of the following benefits:

(iv) Increased transfection efficiency compared to reference LNP; and

52. The method of claim 50 or 51, wherein the modulation of cellular function comprises reprogramming the immune cells to initiate an immune response.

53. The method of claim 50 or 51, wherein the modulation of cellular function comprises modulating antigen specificity of the immune cells.

54. A method of treating, ameliorating, or preventing a symptom of a disorder or disease in a subject in need thereof, the method comprising administering to the subject Lipid Nanoparticles (LNPs) to deliver nucleic acids to immune cells of the subject, wherein the LNPs comprise:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the nucleic acid modulates an immune response of the immune cell, thereby treating or ameliorating the symptom.

55. The method of claim 50 wherein the LNP provides at least one of the following benefits:

(iii) Increased transfection efficiency compared to reference LNP;

(v) The LNP can be administered at a lower dose than the reference LNP to achieve the same therapeutic efficacy; and

56. The method of claim 54 or 55, wherein the disorder is an immune disorder, an inflammatory disorder, or cancer.

57. The method of claim 54 or 55, wherein the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine for treating or preventing a pathogen infection.

58. The method of any one of claims 44-57, wherein the ionizable cationic lipid is

59. The method of any one of claims 44 to 57, wherein the immune cell targeting moiety comprises an antibody that binds a T cell antigen.

60. The method of claim 57, wherein the T cell antigen is CD3, CD8, or both CD3 and CD 8.

61. The method of any one of claims 44 to 56, wherein the immune cell targeting group comprises an antibody that binds a Natural Killer (NK) cell antigen.

62. The method of claim 60, wherein the NK cell antigen is CD7, CD8, or CD56.

63. The method of any one of claims 58-61, wherein the antibody is a human or humanized antibody.

64. The method of any one of claims 44-62, wherein the immune cell targeting group is covalently coupled to a lipid in the lipid blend via a linker comprising polyethylene glycol (PEG).

65. The method of claim 63, wherein the lipid covalently coupled to the immune cell targeting group via a PEG-containing linker is distearoyl glycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycerol-phosphate glycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (dpp), or ceramide.

66. The method of claim 63 or 64, wherein the PEG is PEG 2000.

67. The method of any one of claims 46 to 65, wherein the lipid-immune cell targeting group conjugate is present in the lipid blend in a range of 0.002-0.2 mole percent.

68. The method of any one of claims 46 to 66, wherein the ionizable cationic lipid is present in the lipid blend in a range of 40-60 mole percent.

69. The method of claims 44-67, wherein the sterol is cholesterol.

70. The method of any one of claims 44-68, wherein the sterol is present in the lipid blend in a range of 30-50 mole percent.

71. The method of claims 44-69, wherein the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM).

72. The method of claims 44-70, wherein the neutral phospholipid is present in the lipid blend in a range of 1-10 mole percent.

73. The method of any one of claims 44-71, wherein the free PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, the PEG lipid may be PEG-dioleoyl glycerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-dppg), PEG-diiodoyl-glycerol-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoyl glycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycerol-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycerol-phosphoethanolamine (PEG-DOPE), 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide, or PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

74. The method of claims 44-71, wherein the free PEG-lipid comprises a diacyl phosphatidylethanolamine comprising a dipalmitoyl (C16) chain or a distearoyl (C18) chain.

75. The method of any one of claims 44-73, wherein the free PEG-lipid is present in the lipid blend in a range of 2-4 mole percent.

76. The method of any one of claims 65-74, wherein the free PEG-lipid comprises the same or different lipid as the lipid in the lipid-immune cell targeting group conjugate.

77. The method of claims 44-73 wherein the LNP has an average diameter in the range of 50-200 nm.

78. The method of claim 74 wherein the LNP has an average diameter of about 100 nm.

79. The method of claims 44-77 wherein said LNP has a polydispersity index in the range of from 0.05 to 1.

80. The method of claims 44-78 wherein the LNP has a zeta potential of from about-10 mV to about +30mV at pH 5.

81. The method of claims 44-79, wherein the nucleic acid is DNA or RNA.

82. The method of claim 80, wherein the RNA is mRNA, tRNA, siRNA or microrna.

83. The method of claim 81, wherein the mRNA encodes a receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen, an enzyme, or a vaccine.

84. The method of claim 81, wherein the mRNA encodes a polypeptide capable of modulating an immune response in the immune cell.

85. The method of claim 81, wherein the mRNA encodes a polypeptide capable of reprogramming the immune cell.

86. The method of claim 81, wherein the mRNA encodes a synthetic T cell receptor (synTCR) or Chimeric Antigen Receptor (CAR).

87. The method of any one of claims 44-85, wherein the immune cell targeting group comprises an antibody, and the antibody is a Fab or immunoglobulin single variable domain.

88. The method of any one of claims 44-85, wherein the immune cell targeting group comprises an antibody fragment selected from the group consisting of: fab, F (ab ') 2, fab' -SH, fv and scFv fragments.

89. The method of claim 86 or 87, wherein the immune cell targeting group comprises a Fab containing one or more interchain disulfide bonds.

90. The method of claim 88, wherein the Fab comprises a heavy chain fragment comprising F174C and C233S substitutions and a light chain fragment comprising S176C and C214S substitutions, numbered in accordance with Kabat.

91. The method of any one of claims 86-89, wherein the immune cell targeting group comprises a Fab containing a cysteine at the C-terminus of the heavy or light chain fragment.

92. The method of claim 86, wherein the Fab further comprises one or more amino acids between the heavy chain fragment of the Fab and the C-terminal cysteine.

93. The method of any one of claims 87-91, wherein the Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and wherein the immune cell targeting group further comprises a single chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain via an amino acid linker.

94. The method of claim 86, wherein the immune cell targeting group comprises an immunoglobulin single variable domain.

95. The method of claim 86 or 93, wherein the immunoglobulin single variable domain comprises a cysteine at the C-terminus.

96. The method of claim 94, wherein the immunoglobulin single variable domain comprises a VHH domain and further comprises a spacer comprising one or more amino acids between the VHH domain and the C-terminal cysteine.

97. The method of any one of claims 86 and 93-95, wherein the immune cell targeting group comprises two or more VHH domains.

98. The method of claim 96, wherein the two or more VHH domains are connected by an amino acid linker.

99. The method of claim 96, wherein the immune cell targeting group comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain, and wherein the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds.

100. The method of any one of claims 86 and 93-95, wherein the immune cell targeting group comprises a VHH domain linked to an antibody CH1 domain, and wherein the antibody CH1 domain is linked to an antibody light chain constant domain by one or more disulfide bonds.

101. The method of claim 96 or 97, wherein the CH1 domain comprises F174C and C233S substitutions and the light chain constant domain comprises S176C and C214S substitutions, numbered according to Kabat.

102. The method of any one of claims 44-85, wherein the immune cell targeting group comprises a Fab comprising:

103. The method of any one of claims 44-101, wherein no more than 5% of non-immune cells are transfected with the LNP.

104. The method of any one of claims 44-102, wherein the half-life of the nucleic acid delivered by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP is at least 10% longer than the half-life of the nucleic acid delivered by a reference LNP or the polypeptide encoded by the nucleic acid delivered by the reference LNP.

105. The method of any one of claims 44-103, wherein at least 10% of immune cells are transfected with the LNP.

106. The method of any one of claims 44-104, wherein the expression level of the nucleic acid delivered by the LNP is at least 10% higher than the expression level of a nucleic acid delivered by a reference LNP.

107. A Lipid Nanoparticle (LNP) for delivering a nucleic acid to an immune cell of a subject, wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the immune cell is an NK cell and the immune cell targeting group comprises an antibody that binds CD 56.

108. A Lipid Nanoparticle (LNP) for delivering a nucleic acid to an immune cell of a subject, wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the immune cell targeting group comprises an antibody that binds CD7 or CD8 and the free PEG lipid is DMG-PEG.

109. A Lipid Nanoparticle (LNP) for delivering a nucleic acid to an immune cell of a subject, wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the immune cell targeting group comprises an antibody and the antibody is a Fab or immunoglobulin single variable domain.

110. The LNP of claim 108, wherein the Fab is engineered to knock out the natural interchain disulfide bond at the C-terminus.

111. The LNP of claim 109, wherein said Fab comprises a heavy chain fragment comprising a C233S substitution and a light chain fragment comprising a C214S substitution.

112. The LNP of claim 110, wherein said Fab comprises a non-natural interchain disulfide linkage.

113. The LNP of claim 110, wherein the Fab comprises an F174C substitution in the heavy chain fragment and an S176C substitution in the light chain fragment.

114. The LNP of claim 108, wherein the antibody is an Immunoglobulin Single Variable (ISV) domain and the ISV domain isISV。

115. The LNP of claim 113, wherein said free PEG lipid comprises PEG having a molecular weight of at least 2000 daltons.

116. The LNP of claim 114, wherein said PEG has a molecular weight of about 3000 to 5000 daltons.

117. The LNP of claim 108, wherein the antibody is a Fab.

118. The LNP of claim 116, wherein the Fab binds to CD3 and the free PEG lipid in the LNP comprises PEG having a molecular weight of about 2000 daltons.

119. The LNP of claim 116, wherein the Fab is an anti-CD 4 antibody and the free PEG lipid in the LNP comprises PEG having a molecular weight of about 3000 to 3500 daltons.

120. The LNP of claim 108, wherein the immune cell targeting group comprises two or more VHH domains.

121. The LNP of claim 119, wherein the two or more VHH domains are linked by an amino acid linker.

122. The LNP of claim 120, wherein the immune cell targeting group comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain.

123. A Lipid Nanoparticle (LNP) for delivering a nucleic acid to an immune cell of a subject, wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

the LNP binds to CD3 and also to CD11a or CD18.

124. The LNP of claim 122, wherein the LNP comprises two conjugates, wherein a first conjugate comprises an antibody that binds CD3 and a second conjugate comprises an antibody that binds CD11a or CD18.

125. The LNP of claim 122, wherein the LNP comprises a conjugate and the conjugate comprises a binding CD3 andCD11abispecific antibodies of both.

126. The LNP of claim 122 wherein the LNP comprises a conjugate and the conjugate comprises a bispecific antibody that binds both CD3 and CD18.

127. The LNP of claim 124 or 125, wherein the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

128. A Lipid Nanoparticle (LNP) for delivering a nucleic acid to an immune cell of a subject, wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the LNP binds to CD7 and CD8 of the immune cell.

129. The LNP of claim 127, wherein the LNP comprises two conjugates, wherein a first conjugate comprises an antibody that binds CD7 and a second conjugate comprises an antibody that binds CD8.

130. The LNP of claim 127 wherein the LNP comprises a conjugate, wherein the conjugate comprises bispecific antibodies that bind CD7 and CD8.

131. The LNP of claim 129, wherein the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

132. A Lipid Nanoparticle (LNP) for delivering nucleic acid to two different types of immune cells of a subject, wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the LNP binds to a first antigen on the surface of the immune cell of the first type and also binds to a second antigen on the surface of the immune cell of the second type.

133. The LNP of claim 131, wherein the two different types of immune cells are cd4+ T cells and cd8+ T cells.

134. The LNP of claim 131 wherein the LNP comprises two conjugates and a first conjugate comprises a first antibody that binds to the first antigen of the first type of immune cell and a second conjugate comprises a second antibody that binds to the second antigen of the second type of immune cell.

135. The LNP of claim 131 wherein the LNP comprises a conjugate and the conjugate comprises a bispecific antibody and the bispecific antibody binds to both the first antigen on the first type of immune cell and the second antigen on the second type of immune cell.

136. The LNP of claim 134, wherein the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

137. A Lipid Nanoparticle (LNP) for delivering nucleic acid to both cd4+ and cd8+ T cells of a subject, wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the immune cell targeting group comprises a single antibody that binds to CD3 or CD 7.

138. A Lipid Nanoparticle (LNP) for delivering a nucleic acid to both T cells and NK cells of a subject, wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the immune cell targeting moiety binds to CD7, CD8 or both CD7 and CD 8.

139. A Lipid Nanoparticle (LNP) for delivering a nucleic acid to both T cells and NK cells of a subject, wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the immune cell targeting group binds to:

(i) Both CD3 and CD 56;

(ii) Both CD8 and CD 56; or alternatively

(iii) Both CD7 and CD 56.

140. The LNP of any one of claims 106-138, wherein the immune cell targeting group is covalently coupled to a lipid in the lipid blend via a linker comprising polyethylene glycol (PEG).

141. The LNP of claim 139, wherein the lipid that is covalently coupled to the immune cell targeting group via a PEG-containing linker is distearoyl glycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycerol-phosphate glycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (dpp), or ceramide.

142. The LNP of any one of claims 106-130, wherein the lipid-immune cell targeting group conjugate is present in the lipid blend in a range of 0.002-0.2 mole percent.

143. The LNP of any one of claims 106-141, wherein the lipid blend further comprises one or more of a structural lipid (e.g., a sterol), a neutral phospholipid, and a free PEG-lipid.

144. The LNP of any one of claims 106-142, wherein the ionizable cationic lipid is present in the lipid blend in a range of 40-60 mole percent.

145. The LNP of claim 142, wherein the sterol is present in the lipid blend in a range of 30-50 mole percent.

146. The LNP of claim 142 or 144, wherein the sterol is cholesterol.

147. An LNP according to any one of claims 138 to 145 wherein the neutral phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

148. An LNP according to any one of claims 106 to 146, wherein the neutral phospholipid is present in the lipid blend in a range of 1-10 mole percent.

149. The LNP of any one of claims 106-146, wherein the free PEG-lipid is selected from PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-distearoyl-phosphatidylethanolamine (PEG-DMPE), N- (methylpolyoxycarbonyl) -1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1, 2-dimyristoyl-rac-glycerol-3-methylpolyethylene oxide (PEG-DMG), 1, 2-dipalmitoyl-rac-glycerol-3-methylpolyethylene oxide (PEG-dpp), 1, 2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG), 1, 2-distearoyl-rac-glycerol-3-methylpolyethylene oxide (PEG-DSPE), N-palmitoyl-sphingosine-1- { succinyl [ methoxy (polyethylene glycol) ] (PEG-ceramide), and DSPE-PEG-cysteine, or derivatives thereof.

150. The LNP of any one of claims 106-148, wherein the free PEG-lipid comprises a diacyl phosphatidylethanolamine comprising a dipalmitoyl (C16) chain or a distearoyl (C18) chain.

151. The LNP of any one of claims 106-149, wherein the free PEG-lipid is present in the lipid blend in a range of 1-2 mole percent.

152. The LNP of any one of claims 106-150, wherein the free PEG-lipid comprises the same or different lipid as the lipid in the lipid-immune cell targeting group conjugate.

153. An LNP according to any one of claims 106 to 151 wherein the LNP has an average diameter in the range of 50-200 nm.

154. The LNP of claim 152 wherein the LNP has an average diameter of about 100 nm.

155. The LNP of any one of claims 106-153, wherein the LNP has a polydispersity index in a range from 0.05 to 1.

156. The LNP of any one of claims 106-154 wherein the LNP has a zeta potential of from about-10 mV to about +30mV at pH 5.

157. The LNP of any one of claims 106-155, wherein the nucleic acid is DNA or RNA.

158. The LNP of claim 156, wherein the RNA is mRNA.

159. The LNP of claim 157, wherein the mRNA encodes a receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen, an enzyme, or a vaccine.

160. The LNP of claim 157, wherein the mRNA encodes a polypeptide capable of modulating an immune response in the immune cell.

161. The LNP of claim 159, wherein the mRNA encodes a polypeptide capable of reprogramming the immune cell.

162. The LNP of claim 160, wherein the mRNA encodes a synthetic T cell receptor (synTCR) or a Chimeric Antigen Receptor (CAR).

163. A Lipid Nanoparticle (LNP) for delivering a nucleic acid to an immune cell of a subject, wherein the LNP comprises:

(a) An ionizable cationic lipid is present in the composition,

(b) A conjugate comprising the structure:

[ lipid ] - [ optional linker ] - [ immune cell targeting group ];

(c) Sterols or other structural lipids;

(d) Neutral phospholipids;

(e) Free polyethylene glycol (PEG) lipids, and

(f) The nucleic acid sequence of the nucleic acid sequence,

wherein the immune cell targeting group comprises a Fab lacking native interchain disulfide bonds.

164. The LNP of claim 162, wherein the Fab is engineered to replace one or both cysteines on the natural constant light chain and natural constant heavy chain that form natural inter-chain disulfide bonds with non-cysteine amino acids, thereby removing natural inter-chain disulfide bonds in the Fab.

165. A method of targeting delivery of a nucleic acid to an immune cell of a subject, the method comprising contacting the immune cell with a Lipid Nanoparticle (LNP) of any one of claims 106 to 163.

166. The method of claim 164, wherein the method is for targeting NK cells, wherein the immune cell targeting moiety binds to CD 56.

167. The method of claim 164, wherein the method is for targeting both T cells and NK cells simultaneously, wherein the immune cell targeting moiety binds to CD7, CD8, or both CD7 and CD 8.

168. The method of claim 164, wherein the method is for simultaneously targeting both cd4+ and cd8+ T cells, wherein the immune cell targeting moiety comprises a polypeptide that binds to CD3 or CD 7.

169. A method of expressing a polypeptide of interest in a targeted immune cell of a subject, the method comprising contacting the immune cell with a Lipid Nanoparticle (LNP) of any one of claims 106-163.

170. A method of modulating cellular function of a target immune cell in a subject, the method comprising administering to the subject the Lipid Nanoparticle (LNP) of any one of claims 106 to 159.

171. A method of treating, ameliorating or preventing a symptom of a disorder or disease in a subject in need thereof, the method comprising administering to the subject the Lipid Nanoparticle (LNP) of any one of claims 106-163.

172. An Immunoglobulin Single Variable Domain (ISVD) that binds to human CD8, wherein said ISVD comprises three complementarity determining domains CDR1, CDR2 and CDR3, wherein

(a) The CDR1 comprises GSTFSDYG (SEQ ID NO: 100),

(b) The CDR2 comprises an IDWNGEHT (SEQ ID NO: 101), an

(c) The CDR3 includes AADALPYTVRKYNY (SEQ ID NO: 102).

173. The ISVD of claim 171, wherein said ISVD is humanized.

174. The ISVD of claim 172, wherein said ISVD comprises SEQ ID NO 77.

175. A polypeptide comprising GSTFSDYG (SEQ ID NO: 100), idwneht (SEQ ID NO: 101) and AADALPYTVRKYNY (SEQ ID NO: 102).

176. A polypeptide comprising the ISVD according to claim 171.

177. The polypeptide of claim 175, further comprising a second binding moiety, wherein the second binding moiety binds to CD8 or another different target.

178. The polypeptide of claim 176, wherein the second binding moiety is also ISVD.

179. The polypeptide of claim 175, further comprising a detectable label.

180. The polypeptide of claim 175, further comprising a therapeutic agent.

181. A composition comprising the ISVD according to any of claims 171 to 173 or the polypeptide according to any of claims 174 to 179.

182. A pharmaceutical composition comprising the ISVD according to any of claims 171 to 173 or the polypeptide according to any of claims 174 to 179 and a pharmaceutically acceptable carrier.

183. A method of treating a CD 8-related disease or disorder in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 181.

184. The method of claim 182, wherein the disease or disorder is cancer.