Recombinant bivalent monospecific immunoglobulin having at least two variable fragments of heavy chains of an immunoglobulin devoid of light chains

• US 20030088074A1
• Filed: 05/28/2002
• Published: 05/08/2003
• Est. Priority Date: 04/25/1995
• Status: Active Grant

• Current Assignee: Vrije Universiteit Brussel
• Original Assignee: Vrije Universiteit Brussel
• Inventors: Hamers, Raymond, Muyldermans, Serge
• Granted Patent: US 7,655,759 B2
• Time in Patent Office: Days
• US Class Current: 530/387.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/1282 from Clostridium (G)
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/22 from camelids, e.g. camel, llama or dromedary
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/569 Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to fragments, especially variable fragments of immunoglobulins which are by nature devoid of light chains, these fragments being nevertheless capable of exhibiting a recognition and binding activity toward specific antigens. The present invention further relates to the use of such immunoglobulin fragments formed of at least one heavy chain variable fragment or derived therefrom, for therapeutic or veterinary purposes and especially for passive immunotherapy or serotherapy.

Claims

1. Variable fragment (V_HH) of a heavy chain of an immunoglobulin devoid of light chains, which is encoded by a nucleotide sequence obtainable by the following process:

treating blood lymphocytes or other appropriate cells of an animal of the Camelid family previously immunized with a determined antigen, in order to give access to their mRNA,

synthesizing a first strand of cDNA starting from the obtained mRNA,

contacting the obtained cDNA with at least two different primer oligonucleotides in conditions allowing their hybridization to at least two complementary nucleotide sequences contained in the cDNA, said primers comprising a BACK primer (back p1) having the following nucleotide sequence 5′

-GATGTGCAGCTGCAGGCGTCTGG(A/G)GGAGG-3′ and

a FOR primer (forp

     1) replying to the following nucleotide sequence 5′

-CGCCATCAAGGTACCGTTGA-3′

or 5′

-CGCCATCAAGGTACCAGTTGA-3′

amplifying the DNA fragment located between the nucleotide sequence hybridized with said primers and,

recovering amplified DNA corresponding to bands of different size orders including;

a band of around 750 basepairs which is the amplified product of the variable heavy chain (V_H), CH1, hinge and part of CH2 region of a four-chain immunoglobin,

a band of around 620 basepairs which is the amplified product of the variable heavy-chain (V_HH), long hinge, and part of the CH2 of the camel two-chain immunoglobulin IgG2,

a band of around 550 basepairs which is the amplified product of the variable heavy-chain (V_HH), short hinge, and part of the CH2 of the camel two-chain immunoglobulin IgG3,

purifying the two shortest bands from agarose gel, for example by Gene Clean,

recovering the amplified DNA fragments containing nucleotide sequences encoding the V_HH fragments,

digesting the amplified products with restriction enzymes having target sites within the amplified fragments and/or in the nucleotide primers, for example with PstI and BstEII,

recovering the digested amplified DNA fragments,

ligating the amplified DNA fragments to a phasmid vector, for example in a pHEN4 vector, in conditions allowing the expression of the amplified fragments when the obtained recombinant vector is used to transform a host cell,

transforming a determined bacterial host cell for example an E. Coli cell with the obtained recombinant phasmid vector, and growing the cells on selective medium, to form a library,

infecting the obtained library of recombinant host cells after culture in an appropriate selective medium, with bacteriophages, for instance M13K07 bacteriophages to obtain recombinant phagemid virions,

incubating the recombinant host cells in conditions allowing secretion of recombinant phagemid virions particles containing the recombinant phasmid, for instance the pHEN4 phasmid packaged within the Ml 3 virion.

isolating and concentrating the recombinant phagemid virions,

submitting the phagemid virions to several rounds of panning with the antigen of interest previously immobilized, in conditions allowing the adsorption of the phagemid virions on the immobilized antigen,

eluting the adsorbed phagemid virions, and growing them on appropriate cells,

amplifying the phagemid virions by infecting the cells with helper bacteriophage,

recovering the virions and testing them for their binding activity against the antigen of interest, for example by ELISA,

recovering the phagemid virions having the appropriate binding activity,

isolating the nucleotide sequence contained in the phasmid vector and capable of being expressed on the phagemid virions as a V_HH aminoacid sequence having the appropriate binding activity.

2. Variable fragment (V_HH) of a heavy chain of an immunoglobulin, which is encoded by a nucleotide sequence obtainable by a process according to the one disclosed in claim 1, wherein a reamplification step of the 620 basepairs and 550 basepairs PCR product of claim 1 performed with oligonucleotide primers having respectively the following nucleotide sequences:

BACK primer;

5′

-GATGTGCAGCTGCAGGCGTCTGG(A/G)GGAGG-3′

FOR primer;

5′

- CG ACT AGT GCG GCC GCG TGA GGA GAC GGT GAC CTG-3′

3. Variable fragment (V_HH) of a heavy chain of an immunoglobulin, which is encoded by a nucleotide sequence obtainable by a process according to the one disclosed in claim 1, wherein the amplification step of the cDNA obtained from the mRNA is performed with oligonucleotide primers having respectively the following nucleotide sequences:

BACK primer;

5′

-GATGTGCAGCTGCAGGCGTCTGG(A/G)GGAGG-3′

FOR primer 3;

5′

-TGT CTT GGG TTC TGA GGA GAC GGT -3′

FOR primer 4;

5′

- TTC ATT CGT TCC TGA GGA GAC GGT -3′

4. Variable fragment of a heavy chain of an immuglobulin devoid of light chains according to anyone of claims 1 or 2, encoded by a nucleotide sequence obtainable from blood lymphocytes or other appropriate cells of camelids wherein the camelids have been immunized with a determined antigen prior to the treatment of their blood lymphocytes or other appropriate cells.

5. Variable fragment of a heavy chain of an immuglobulin according to anyone of claim 1 to 4 encoded by a nucleotide sequence obtainable from blood lymphocytes or other appropriate cells of camelids characterized in that the camelids have been previously immunized with an antigen which is a toxin of a bacteria or the corresponding toxoid.

6. Variable fragment of a heavy chain of an immuglobulin according to claim 5 encoded by a nucleotide sequence obtainable from blood lymphocytes or other appropriate cells of camelids wherein the antigen is the tetanus toxoid of Clostridium tetani.

7. Variable fragment of a heavy chain of an immuglobulin encoded by a nucleotide sequence according to claim 5 wherein the antigen is a bacterial toxin or toxoid chosen among those of the following bacteria:

Clostridium, especially Clostridium Botulinum or Clostridium Perfringens, Staphylococcus, Pseudomonas, Pasteurella, Yersinia, Bacillus Anthracis, Neisseria, Vibrio, especially Vibrio cholera, enterotoxic E. Coli, Salmonella, Shigella, Listeria.

8. Variable fragment of a high chain of an immuglobulin according to anyone of claims 1 to 4 encoded by a nucleotide sequence obtainable from blood lymphocytes or other appropriate cells of camelids, wherein the camelids have been immunized with an antigen present in venom of animals.

9. Variable fragment of a high chain of an immuglobulin according to claim 8, encoded by a nucleotide sequence wherein the antigen is a toxin or toxoid chosen among those produced by anemonies, coral, jellyfish, spiders, beas, wasps, scorpions, snakes, including those belonging to the families of Viperidae, Crotalidae, Lapidea.

10. Variable fragment of a heavy chain immunoglobulin encoded by a nucleotide sequence obtained by a process of anyone of claims 1 to 9.

16. Variable fragment of a heavy chain of an immunoglobulin according to anyone of claims 1 to 15 characterized in that it is linked to at least one further variable fragment of heavy chains of an immunoglobulin devoid of light chains according to anyone of claims 1 to 15, the V_HH fragments having the same antigen specificity.

17. Bivalent monospecific construct of variable fragments of an immunoglobulin according to claim 16, which is a dimer of V_HH fragments having the same specificity.

18. Bivalent monospecific construct according to claim 17, wherein the V_HH fragments are linked to each other with the hinge amino-acid sequence of the hinge domain of an immunoglobulin devoid of light chain.

19. Variable fragment of a heavy chains of an immunoglobulin according to anyone of claims 1 to 15 characterized in that it is linked to at least one further variable fragment of heavy chains of an immunoglobulin devoid of light according to anyone of claims 1 to 15, the V_HH fragments having different antigen specificities.

20. Multivalent multispecific construct wherein variable fragments according to claim 19 are linked to each other with part of the amino-acid sequence of the hinge domain of an immunoglobulin devoid of light chains, this part being devoid of at least the codon encoding a cysteine residue at the end of the hinge domain.

21. Multivalent multispecific construct according to claim 20, which contains at least two V_HH fragments having a different antigen- and/or epitope-specificity.

22. Construct according to anyone of claims 16 to 18 wherein the sequence of the the hinge domain includes or corresponds to the amino-acid sequence starting at position 400 and ending between position 489 and position 495 as indicated on the sequence of FIG. 15.

23. Construct according to anyone of claims 19 to 22 wherein the sequence of part of the hinge region includes or corresponds to the amino-acid sequence starting at position 400 and ending between position 479 and position 486 as indicated on the sequence of FIG. 15.

24. Pharmaceutical composition, characterized in that it comprises an immunoglobulin variable fragment according to anyone of claims 1 to 6 or 19 or a construct according to anyone of claims 17, 18, 20 to 23 in admixture with a physiologically acceptable vehicle and/or adjuvant(s).

25. Pharmaceutical composition according to claim 16 for the treatment by passive immunisation, of infection or acute intoxication by toxins such as those of Clostridium, especially Clostridium Botulinum or Clostridium Perfrinaens, Staphylococcus, Pseudomonas, Pasteurella, Yersinia, Bacillus AnthracisNeisseria, Vibrio, especially Vibrio cholera, enterotoxic E. Coli, Salmonella, Shigella, Listeria or anemonies, coral, jellyfish, spiders, beas, wasps, scorpions, snakes, including those belonging to the families of Viperidae, Crotalidae, Lapidea.

26. immunoglobulin variable fragment according to anyone of claims 2 to 6 or 19, or a construct according to anyone of claims 17, 18,20 to 23 for use for the treatment by passive immunisation, of infection or acute intoxication by toxins such as those of Clostridium, especially Clostridium Botulinum or Clostridium Perfrinaens, Staphylococcus, Pseudomonas, Pasteurella, Yersinia, Bacillus Anthracis, Neisseria, Vibrio, especially Vibrio cholera, enterotoxic E. Coli, Salmonella, Shigella, Listeria or anemonies, coral, jellyfish, spiders, beas, wasps, scorpions, snakes, including those belonging to the families of Viperidae, Crotalidae, Lapidea.

33. Nucleotide sequence encoding the constructs of anyone of claims 20 to 23.

11. Variable fragment of a heavy chain of an immuglobulin, characterized in that it comprises the following aminoacid sequence:

12. Amino-acid sequence encoded by a nucleotide sequence comprising the sequence starting at nucleotide 400 and ending between nucleotides 479 and 495 of the nucleotides 479 and 495 of the nucleotide sequence presented on FIG. 15.

13. Variable fragment of a heavy chain of an immuglobulin coded by a nucleotide sequence present in recombinant phasmid pHEN4-α

TT2(WK6) deposited at the BCCM/LMBP under accession number LMBP3247.

14. Variable fragment of a heavy chain of an immunoglobulin characterized in that it comprises or it replies to the following α

TT1 sequence;

28. Nucleotide sequence coding for a variable fragment V_HH of a heavy chain of an immunoglobulin devoid of light chain, directed against an epitope of the tetanus toxin of Clostridium tetani, characterized in that it codes for an amino acid sequence according to claim 14 or 15.

32. Expression product in a host cell of a nucleotide sequence according to anyone of claims 28 to 31.

15. Variable fragment of a heavy chain of an immunoglobulin characterized in that it comprises or it replies to the following α

TT2 aminoacid sequence;

27. Nucleotide sequence coding for a variable fragment (V_HH) of a heavy chain of an immunoglobulin obtainable by the following process:

treating blood lymphocytes or other appropriate cells of an animal of the Camelid family previously immunized with a determined antigen, in order to give access to their mRNA,

synthesizing a first strand of cDNA starting from the obtained mRNA,

contacting the obtained cDNA with at least two different oligonucleotide primers in conditions allowing their hybridization to at least two complementary nucleotide sequences contained in the cDNA, said primers comprising a BACK primer (back p1) having the following nucleotide sequence 5′

-GATGTGCAGCTGCAGGCGTCTGG(A/G)GGAGG-3′ and

a FOR primer (forp

     1) replying to the following nucleotide sequence 5′

-CGCCATCAAGGTACCGTTGA-3′

or 5′

-CGCCATCAAGGTACCAGTTGA-3′

amplifying the DNA fragment located between the nucleotide sequence hybridized with said primers and,

recovering amplified DNA corresponding to bands of different size orders including;

a band of around 750 basepairs which is the amplified product of the variable heavy chain (V_H), CH1, hinge and part of CH2 region of a four-chain immunoglobin,

a band of around 620 basepairs which is the amplified product of the variable heavy-hain (V_HH), long hinge, and part of the CH2 of the camel two-chain immunoglobulin IgG2,

a band of around 550 basepairs which is the amplified product of the variable heavy-chain (V_HH), short hinge, and part of the CH2 of the camel two-chain immunoglobulin IgG3,

purifying the two shortest bands of 620 and 550 basepairs from agarose gel, for example by Gene Clean,

recovering the amplified DNA fragments containing nucleotide sequences encoding the V_HH fragments,

digesting the amplified products with restriction enzymes having target sites within the amplified fragments and/or in the nucleotide primers, for example with PstI and BstEII,

recovering the digested amplified DNA fragments,

ligating the amplified DNA fragments to a phasmid vector, for example in a pHEN4 vector, in conditions allowing the expression of the amplified fragments when the obtained recombinant vector is used to transform a host cell,

transforming a determined bacterial host cell for example an E. Coli cell with the obtained recombinant phasmid vector, and growing the cells on selective medium, to form a library,

infecting the obtained library of recombinant host cells after culture in an appropriate selective medium, with bacteriophages, for instance M13K07 bacteriophages to obtain recombinant phagemid virions,

harvesting the recombinant host cells, adsorbed with the bacteriophages,

incubating the recombinant host cells in conditions allowing secretion of recombinant phagemid virions particles containing the recombinant phasmid, for instance the pHEN4 phasmid packaged within the Ml 3 virion.

isolating and concentrating the recombinant phagemid virions,

submitting the phagemid virions to several rounds of panning with the antigen of interest previously immobilized, in conditions allowing the adsorption of the phagemid virions on the immobilized antigen,

eluting the adsorbed phagemid virions, and growing them on appropriate cells,

amplifying the phagemid virions by infecting the cells with helper bacteriophage,

recovering the virions and testing them for their binding activity against the antigen of interest, for example by ELISA,

recovering the phagemid virions having the appropriate binding activity,

isolating the nucleotide sequence contained in the phasmid vector and capable of being expressed on the phagemid virions as a V_HH aminoacid sequence having the appropriate binding activity.

30. Nucleotide sequence starting with nucleotide 440 and ending between nucleotide 489 and nucleotide 495 on the nucleotide sequence of FIG. 15 or this nucleotide sequence in combination with a nucleotide sequence according to anyone of claims 27 or 28.

31. Nucleotide sequence starting with nucleotide 440 and ending between nucleotide 479 and nucleotide 486 on the nucleotide sequence of FIG. 15 or this nucleotide sequence in combination with a nucleotide sequence according to anyone of claims 27 or 28.

29. Nucleotide sequence coding for a variable fragment V_HH of a heavy chain of an immunoglobulin devoid of light chain, directed against an epitope of the tetanus toxin of Clostridium tetani, characterized in that is comprises one of the following nucleotide sequences:

34. Process for the preparation of monovalent bispecific DNA constructs encoding variable fragments of a heavy chain of an immunoglobulins which comprises the following steps:

a) ligating a nucleotide sequence coding for a variable V_HH fragment having a determined antigen- or epitope- specificity to a linker nucleotide sequence to form a V_HH-linker fragment;

b) ligating the formed nucleotide sequence coding for the V_HH-linker fragment to a nucleotide sequence coding for another V_HH fragment having a different antigen- and/or epitope-specificity,

wherein the linker sequence contains the nucleotide sequence coding for part of a hinge domain wherein the codons responsible for the dimerisation of the V_HH fragments especially by formation of a disulfide bridge between the last cysteine residues within the hinge domain are deleted.

35. Process according to claim 34 comprising one or several additional step(s) of ligation

36. Process according to claim 34 or 35 wherein the sequence encoding part of the hinge domain comprises or corresponds to the nucleotide sequence starting with nucleotide 400 and ending with one of the nucleotides between nucleotide 479 and 486 of the nucleotide sequence on the sequence of FIG. 15.