PROTEINS COMPRISING A MUTATED LAIR-1 FRAGMENT AND USES THEREOF

Abstract

The present invention provides a protein comprising a mutated LAIR-1 fragment, which broadly binds to erythrocytes infected with Plasmodium falciparum. The protein comprising the mutated LAIR-1 fragment may be useful in the prevention and/or treatment of malaria. The present invention furthermore provides a nucleic acid encoding a mutated LAIR-1 fragment, a vector comprising such a nucleic acid as well as a respective pharmaceutical composition.

Claims

1.-74. (canceled)

75. A protein comprising at least amino acids 67 to 107 of native human LAIR-1, wherein said LAIR-1 fragment comprises: i) 1, 2, 3, 4, or 5 mutations in comparison to native human LAIR-1 at one or more of the following five positions: T67, N69, A77, P106, and P107; and ii) optionally, one or more further mutations at a position different from T67, N69, A77, P106, and P107 in comparison to native human LAIR-1, wherein said LAIR-1 fragment has at least 70% amino acid sequence identity to amino acids 67 to 107 of native human LAIR-1 (SEQ ID NO: 9).

76. The protein according to claim 75 comprising at least the mutation N69S and/or T67L.

77. The protein according to claim 75, wherein said LAIR-1 fragment includes a mutation in at least two of the following five positions: T67, N69, A77, P106, and P107.

78. The protein according to claim 75, wherein said LAIR-1 fragment includes a mutation at each of the following five positions: T67, N69, A77, P106, and P107.

79. The protein according to claim 77 or 78, wherein the threonine residue at position T67 is substituted by an amino acid selected from the group consisting of leucine, glycine, isoleucine, arginine and lysine.

80. The protein according to claim 77 or 78, wherein the asparagine residue at position N69 is substituted by an amino acid selected from the group consisting of serine and threonine.

81. The protein according to claim 77 or 78, wherein the alanine residue at position A77 is substituted by an amino acid selected from the group consisting of threonine, proline and valine.

82. The protein according to claim 77 or 78, wherein the proline residue at position P106 is substituted by an amino acid selected from the group consisting of serine, alanine and aspartic acid.

83. The protein according to claim 77 or 78, wherein the proline residue at position P107 is substituted by an amino acid selected from the group consisting of serine and arginine.

84. The protein according to claim 75 comprising the LAIR-1 fragment according to SEQ ID NO: 19, 20, 21 or 22.

85. The protein according to claim 75, wherein the LAIR-1 fragment comprises at least the following mutations in comparison to native human LAIR-1: (i) T67L and A77V or A77T, and/or (ii) T67L, N69S, A77T, P106S, and P107R.

86. The protein according to claim 75, wherein the protein binds to a Plasmodium falciparum surface antigen.

87. The protein according to claim 75, wherein the protein neutralizes infection by Plasmodium falciparum.

88. The protein according to claim 75, wherein the protein does not bind collagen.

89. The protein according to claim 75 comprising a LAIR-1 fragment having an amino acid sequence according to any of SEQ ID NOs: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 and 103 or according to a functional sequence variant thereof.

90. The protein according to claim 75, wherein the protein is an antibody.

91. The protein according to claim 90, wherein the protein comprises a heavy chain comprising the amino acid sequence of (i) SEQ ID NO: 120 for CDRH1, SEQ ID NO: 121 for CDRH2 and SEQ ID NO: 122 for CDRH3 or functional sequence variants thereof; (ii) SEQ ID NO: 138 for CDRH1, SEQ ID NO: 139 for CDRH2 and SEQ ID NO: 140 for CDRH3 or functional sequence variants thereof; (iii) SEQ ID NO: 156 for CDRH1, SEQ ID NO: 157 for CDRH2 and SEQ ID NO: 158 for CDRH3 or functional sequence variants thereof; (iv) SEQ ID NO: 174 for CDRH1, SEQ ID NO: 175 for CDRH2 and SEQ ID NO: 176 for CDRH3 or functional sequence variants thereof; (v) SEQ ID NO: 192 for CDRH1, SEQ ID NO: 193 for CDRH2 and SEQ ID NO: 194 for CDRH3 or functional sequence variants thereof; (vi) SEQ ID NO: 210 for CDRH1, SEQ ID NO: 211 for CDRH2 and SEQ ID NO: 212 for CDRH3 or functional sequence variants thereof; (vii) SEQ ID NO: 228 for CDRH1, SEQ ID NO: 229 for CDRH2 and SEQ ID NO: 330 for CDRH3 or functional sequence variants thereof; (viii) SEQ ID NO: 246 for CDRH1, SEQ ID NO: 247 for CDRH2 and SEQ ID NO: 248 for CDRH3 or functional sequence variants thereof; (ix) SEQ ID NO: 264 for CDRH1, SEQ ID NO: 265 for CDRH2 and SEQ ID NO: 266 for CDRH3 or functional sequence variants thereof; (x) SEQ ID NO: 282 for CDRH1, SEQ ID NO: 283 for CDRH2 and SEQ ID NO: 284 for CDRH3 or functional sequence variants thereof; (xi) SEQ ID NO: 300 for CDRH1, SEQ ID NO: 301 for CDRH2 and SEQ ID NO: 302 for CDRH3 or functional sequence variants thereof; (xii) SEQ ID NO: 318 for CDRH1, SEQ ID NO: 319 for CDRH2 and SEQ ID NO: 320 for CDRH3 or functional sequence variants thereof; (xiii) SEQ ID NO: 336 for CDRH1, SEQ ID NO: 337 for CDRH2 and SEQ ID NO: 338 for CDRH3 or functional sequence variants thereof; (xiv) SEQ ID NO: 354 for CDRH1, SEQ ID NO: 355 for CDRH2 and SEQ ID NO: 356 for CDRH3 or functional sequence variants thereof; (xv) SEQ ID NO: 372 for CDRH1, SEQ ID NO: 373 for CDRH2 and SEQ ID NO: 374 for CDRH3 or functional sequence variants thereof; (xvi) SEQ ID NO: 390 for CDRH1, SEQ ID NO: 391 for CDRH2 and SEQ ID NO: 392 for CDRH3 or functional sequence variants thereof; (xvii) SEQ ID NO: 408 for CDRH1, SEQ ID NO: 409 for CDRH2 and SEQ ID NO: 410 for CDRH3 or functional sequence variants thereof; (xviii) SEQ ID NO: 426 for CDRH1, SEQ ID NO: 427 for CDRH2 and SEQ ID NO: 428 for CDRH3 or functional sequence variants thereof; (xix) SEQ ID NO: 444 for CDRH1, SEQ ID NO: 445 for CDRH2 and SEQ ID NO: 446 for CDRH3 or functional sequence variants thereof; (xx) SEQ ID NO: 462 for CDRH1, SEQ ID NO: 463 for CDRH2 and SEQ ID NO: 464 for CDRH3 or functional sequence variants thereof; (xxi) SEQ ID NO: 480 for CDRH1, SEQ ID NO: 481 for CDRH2 and SEQ ID NO: 482 for CDRH3 or functional sequence variants thereof; (xxii) SEQ ID NO: 498 for CDRH1, SEQ ID NO: 499 for CDRH2 and SEQ ID NO: 500 for CDRH3 or functional sequence variants thereof; (xxiii) SEQ ID NO: 516 for CDRH1, SEQ ID NO: 517 for CDRH2 and SEQ ID NO: 518 for CDRH3 or functional sequence variants thereof; (xxiv) SEQ ID NO: 534 for CDRH1, SEQ ID NO: 535 for CDRH2 and SEQ ID NO: 536 for CDRH3 or functional sequence variants thereof; (xxv) SEQ ID NO: 552 for CDRH1, SEQ ID NO: 553 for CDRH2 and SEQ ID NO: 554 for CDRH3 or functional sequence variants thereof; or (xxvi) SEQ ID NO: 570 for CDRH1, SEQ ID NO: 571 for CDRH2 and SEQ ID NO: 572 for CDRH3 or functional sequence variants thereof.

92. The protein according to claim 90, wherein the protein comprises (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 134 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 135 or a functional sequence variant thereof; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 152 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 153 or a functional sequence variant thereof; or (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 170 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 171 or a functional sequence variant thereof; or (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 188 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 189 or a functional sequence variant thereof; or (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 206 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 207 or a functional sequence variant thereof; or (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 224 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 225 or a functional sequence variant thereof; or (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 242 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 243 or a functional sequence variant thereof; or (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 260 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 261 or a functional sequence variant thereof; or (ix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 278 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 279 or a functional sequence variant thereof; or (x) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 296 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 297 or a functional sequence variant thereof; or (xi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 314 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 315 or a functional sequence variant thereof; or (xii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 332 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 333 or a functional sequence variant thereof; or (xiii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 350 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 351 or a functional sequence variant thereof; or (xiv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 368 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 369 or a functional sequence variant thereof; or (xv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 386 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 387 or a functional sequence variant thereof; or (xvi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 404 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 405 or a functional sequence variant thereof; or (xvii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 422 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 423 or a functional sequence variant thereof; or (xviii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 440 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 441 or a functional sequence variant thereof; or (xix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 458 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 459 or a functional sequence variant thereof; or (xx) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 476 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 477 or a functional sequence variant thereof; or (xxi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 494 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 495 or a functional sequence variant thereof; or (xxii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 512 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 513 or a functional sequence variant thereof; or (xxiii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 530 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 531 or a functional sequence variant thereof; or (xxiv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 548 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 549 or a functional sequence variant thereof; or (xxv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 566 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 567 or a functional sequence variant thereof; or (xxvi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 584 or a functional sequence variant thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 585 or a functional sequence variant thereof.

93. A nucleic acid molecule comprising a polynucleotide encoding a protein according to claim 75.

94. A vector comprising the nucleic acid molecule according to claim 93.

95. A cell expressing the protein according to claim 75 or comprising the vector according to claim 94.

96. A LAIR-1 fragment protein comprising at least amino acids 67 to 107 of native human LAIR-1, wherein said LAIR-1 fragment protein comprises at least one mutation in comparison to native human LAIR-1 (SEQ ID NO: 9), said at least one mutation enabling binding to an antigen, and wherein said LAIR-1 fragment shows at least 70% amino acid sequence identity to amino acids 67 to 107 of native human LAIR-1 (SEQ ID NO: 9).

97. The protein according to claim 96, wherein the protein is an antibody, preferably a recombinant antibody.

98. A method of preventing and/or treating a disorder and/or a disease selected from the group consisting of: infectious diseases, autoimmune diseases, inflammatory diseases and cancers in a subject, wherein the method comprises administering to a subject in need thereof the protein according to claim 96.

99. A pharmaceutical composition comprising the protein according to claim 75 and/or the protein according to claim 96, a nucleic acid encoding one or both of said proteins, a vector comprising said nucleic acid, or a cell expressing one or both of said proteins or comprising said vector.

100. A method of diagnosing malaria, comprising contacting a sample suspected of containing a malaria parasite, or portion thereof, with the protein according to claim 75; and detecting a complex of the protein according to claim 75 and the malaria parasite, or portion thereof.

101. A method of limiting a Plasmodium falciparum infection, or lowering the risk of Plasmodium falciparum infection, comprising administering to a subject in need thereof, a therapeutically effective amount of : iii) a protein comprising at least amino acids 67 to 107 of native human LAIR-1, wherein said LAIR-1 fragment comprises: (a) 1, 2, 3, 4, or 5 mutations in comparison to native human LAIR-1 at one or more of the following five positions: T67, N69, A77, P106, and P107; and (b) optionally, one or more further mutations at a position different from T67, N69, A77, P106, and P107 in comparison to native human LAIR-1, wherein said LAIR-1 fragment shows at least 70% amino acid sequence identity to amino acids 67 to 107 of native human LAIR-1 (SEQ ID NO: 9), iv) a nucleic acid encoding said protein, v) a vector comprising said nucleic acid, vi) a cell expressing said protein or comprising said vector, or vii) a pharmaceutical composition comprising said protein and/or the protein according to claim 96, a nucleic acid encoding said protein, a vector comprising said nucleic acid molecule, or a cell expressing said protein or comprising said vector.

102. A method of preventing and/or treating malaria in a subject, wherein the method comprises administering to a subject in need thereof: viii) a protein comprising at least amino acids 67 to 107 of native human LAIR-1, wherein said LAIR-1 fragment comprises: (a) 1, 2, 3, 4, or 5 mutations in comparison to native human LAIR-1 at one or more of the following five positions: T67, N69, A77, P106, and P107; and (b) optionally, one or more further mutations at a position different from T67, N69, A77, P106, and P107 in comparison to native human LAIR-1, wherein said LAIR-1 fragment shows at least 70% amino acid sequence identity to amino acids 67 to 107 of native human LAIR-1 (SEQ ID NO: 9), ix) a nucleic acid encoding said protein, x) a vector comprising said nucleic acid, xi) a cell expressing said protein or comprising said vector, or xii) a pharmaceutical composition comprising said protein and/or the protein according to claim 96, a nucleic acid encoding said protein, a vector comprising said nucleic acid molecule, or a cell expressing said protein or comprising said vector.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0464] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

[0465] FIG. 1 shows for Example 1 an example of staining of P. falciparum-infected erythrocytes by a broadly cross-reactive antibody (MGD21). IEs are stained with SYBR Green I dye (DNA) to discriminate them from uninfected erythrocytes used as control. The graph shows that MGD21 specifically binds only to IEs.

[0466] FIG. 2 shows an alignment of selected monoclonal antibodies of Example 1 (antibodies MGD21, MGD39, MGD47 and MGD55 in FIG. 2A and antibodies MGC1, MGC7, MGC37 and MGC29 in FIG. 2B) to an amino acid sequence encoded by the corresponding fragment of genomic LAIR1 sequence (exon+intron).

[0467] FIG. 3 shows a scheme of the different antibody variants constructed in Example 3. The different elements of the 10 antibody constructs are compared to MGD21 and FI499 (unrelated antibody). MGD21 binds to erythrocytes infected with 9/9 primary P. falciparum isolates and carries the LAIR-1 exon+intron insertion. F1499 is an IgG antibody that binds influenza hemagglutinin and uses different V, D and J elements. D and D indicate two putative D elements. GGGGS is an artificial linker. VH4-4, JH6, VK1-8 and JK5 and LAIR-1 intron and exon were also tested in the germline form (GL). For LAIR-1 the genomic sequence (ENSG00000167613) was used. In the right column, it is indicated whether the antibody (construct) binds to IEs as tested in Example 4.

[0468] FIG. 4 shows the results of Example 4 indicating that the mutated LAIR-1 exon is the only element required for mAb MGD21 binding to P. falciparum-infected erythrocytes. The antibodies were quantitated and tested for their capacity to stain IEs. Con1 refers to FI499_DexinDJ, Con2 refers to FI499VJ_DexinD, Con3 refers to MGD21_exin_longGS, Con4 refers to MGD21_exin_shortGS, Con5 refers to MGD21_NOexin, Con6 refers to MGD21_NOin, Con7 refers to MGD21_NOVD, Con8 refers to MGD21 GL_exinWT, Con9 refers to MGD21_wholeGL.

[0469] FIG. 5 shows a scheme of the different fusion proteins produced in Example 5. M1, M2, M3 and M4 are four different mouse IgG2b fusion proteins comprising the mutated LAIR-1 fragment according to the present invention, while H1 and H2 are two different human IgG1 fusion proteins comprising the mutated LAIR-1 fragment according to the present invention. M1 and H1 share the same variable region. M4 and H2 share the same variable region. D.sub. and D.sub. refer to the expression products of a first fragment and of a second fragment, different from the first fragment, of the same or different D (Diversity) gene segment element of a heavy chain variable region of an IgG-type antibody. JH6 refers to the expression product of a J (Joining) gene segment element of a heavy chain variable region of an IgG-type antibody. Exon refers to the mutated LAIR-1 fragment. Intron and Intron.sub. refer to further LAIR-1 elements (expression products from one LAIR-1 intron fragment, whereby Intron.sub. is a fragment of Intron). Hinge, CH2, and CH3 form together the constant region provided by the plasmid.

[0470] FIG. 6 shows for Example 6 that the mutated LAIR-1 fragment expressed as a fusion protein (cf. Example 5) binds to IEs. The four fusion proteins expressed in the mouse IgG2b fusion-protein vector were quantitated and tested for their capacity to stain IEs.

[0471] FIG. 7 shows for Example 7 that fusion proteins comprising the mutated LAIR-1 fragment efficiently opsonize P. falciparum-infected erythrocytes. Parasites were stained with DAPI and mixed with a titration of antibodies and fusion proteins, followed by incubation with monocytes at 37 C. for 1 hour. Monocytes were stained with anti-CD14-APC and MFI of DAPI (A) and the % of DAPI-positive monocytes (B) were calculated in CD14-positive populations. DexinDJ and exon are two fusion proteins expressed in the human IgG1 vector (cf. Example 5, also referred to as H1 and H2). F1499 is an unrelated antibody used as control. FIG. 7C shows agglutinates of 3D7-MGD21.sup.+ or 11019-MGD21.sup.+ IEs formed by MGD21 or MGC34. Scale bar, 25 m.

[0472] FIG. 8 shows for Example 7 that antibodies MGD21, MG47, MGD55, MGC28 and MGC34 efficiently opsonize P. falciparum-infected erythrocytes. The IEs were stained with 4,6-diamidino-2-phenylindole (DAPI), which was quantified in monocytes as a measure of phagocytosis. (A) Opsonic phagocytosis of 3D7-MGD2.sup.+ IEs by monocytes (n=3 for MGD21, MGD21 LALA and BKC3, n=2 for others). (B) Opsonic phagocytosis of 11019-MGD21.sup.+ IEs by monocytes (n=2).

[0473] FIG. 9 shows for Example 8 an alignment of the mutated LAIR-1 exon of the human monoclonal antibodies of Example 1 with amino acids 24 to 121 of native human LAIR-1 (SEQ ID NO: 14). Positions T67, N69, A77, P106 and P107 are shown in frames.

[0474] FIG. 10 shows for Example 8 the mutated LAIR-1 fragment modeled on the structure of the LAIR-1 extracellular domain. The LAIR-1 structure is shown as cartoon (left) and as surface (right). The five positions, at which a mutation may occur in the mutated LAIR-1 fragment as compared to the native LAIR-1 structure are highlighted in black.

[0475] FIG. 11 shows for Example 9 that the LAIR-1 fragment expressed as a fusion protein and carrying different combinations of mutations at positions T67, N69, A77, P106 and P107 binds to IEs while the same LAIR-1 fragment with no mutations does not bind to IEs. LAIR1 ex is the fusion protein carrying the LAIR1 fragment corresponding to the genomic sequence (Gene: LAIR1 ENSG00000167613). LAIR1ex+X are the fusion protein carrying the LAIR-1 fragment corresponding to the genomic sequence with one or more mutations (only mutated residues [L,S1,T,S2,R] are indicated according to the 5 most preferred mutations respectively: T67L, N695, A77T, P106S, and P107R, whereby L refers to T67L, S1 refers to N69S, T refers to A77T, S2 refers to P106S and R refers to P107R. For instance LAIR1ex+L carries the mutation T67L and LAIR1ex+S2 carries the mutation P106S.

[0476] FIG. 12 shows for Example 10 that the different mutations in the LAIR-1 fragment expressed as a fusion protein and carrying different combinations of mutations at positions T67, N69, A77, P106 and P107 (cf. Example 9) influence binding to collagen. The fusion proteins are the same shown in FIG. 10 (cf. Example 9).

[0477] FIG. 13 depicts a western blot showing MGD21 binding to erythrocyte ghosts and MGD21 immunoprecipitates (IP) prepared from 3D7-MGD21.sup. and 3D7-MGD21.sup. IEs (representative of n=2 independent experiments). Controls include uninfected erythrocytes (uEs) and immunoprecipitates with an irrelevant antibody (BKC3). Specific bands are marked with asterisks. Anti-human IgG was used as the secondary antibody, resulting in detection of antibodies used for immunoprecipitation alongside antigens of interest. Numbers on right indicate kDa.

[0478] FIG. 14 shows a Volcano plot from LC-MS analysis of MGD21 immunoprecipitates prepared from 3D7-MGD21.sup.+ IEs versus from 3D7-MGD21.sup. IEs (from n=4 independent experiments). Statistical significance was evaluated by Welch tests (P<0.01 for PF3 D7_1400600).

[0479] FIG. 15 shows a heat map from LC-MS analysis showing RIFIN expression levels (calculated as intensity-based absolute quantification (iBAQ) scores) in erythrocyte ghosts prepared from 3D7-MGD21.sup.+ and 3D7-MGD21.sup. IEs (two experiments shown). Boxes with crosses indicate that expression levels are below the detection limit.

[0480] FIG. 16 shows the percentage of IEs (representative of n=2 independent experiments) stained by the antibodies. BKC3 is a negative control antibody.

[0481] FIG. 17 shows for Example 11 a western blot (A) showing MGD21 binding to immunoprecipitates (IP) prepared from 9605-MGD21.sup. and 9605-MGD21.sup.+ IEs (representative of n=2 independent experiments). Specific bands are marked with an asterisk. Anti-human IgG was used as the secondary antibody, resulting in detection of antibodies used for immunoprecipitation alongside antigens of interest. FIG. 16B shows percentage of 9605-MGD21.sup. and 9605-MGD21.sup.+ IEs recognized by representative MGC and MGD antibodies (representative of n=2 independent experiments).

[0482] FIG. 18 shows (A) the percentage of transfected CHO cells (n=1) stained by the antibodies. BKC3 is a negative control antibody. FIG. 17B shows MGD21 and BKC3 staining of CHO cells transfected with a specific (PF3D7_1400600) or an irrelevant (PF3D7_0100200) RIFIN (representative of n=5 independent experiments).

[0483] FIG. 19 shows binding of MGD21 (left) or of an Fc fusion protein containing the LAIR1 domain of MGD21 (right) to CHO cells transfected with RIFINs (PF3D7_1400600 and PF3D7_0100200), a RIFIN chimaera containing the constant region of PF3D7_0100200 and the variable region of PF3D7_1400600 (PF3D7_0100200c_1400600v), or the inverse chimaera (PF3D7_1400600c_0100200v) (n=1).

EXAMPLES

[0484] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1

Isolation of Human Monoclonal Antibodies that Broadly React with P. falciparum-Infected Erythrocytes (IE)

[0485] Two African donors (identified as donor C and D) were selected for their high levels of serum antibodies capable of cross-agglutinating erythrocytes infected with different field isolates of P. falciparum. Memory B cells were isolated and immortalized as described by Traggiai, E., et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat. Med. 10, 871-875 (2004) to isolate monoclonal antibodies. Briefly, memory B cells were isolated from cryopreserved PBMCs using anti-FITC microbeads following staining of PBMCs with CD22-FITC, and were immortalized with Epstein-Barr virus and CpG in multiple wells. After 14 days culture supernatants were screened using a high throughput flow cytometer for their capacity to stain infected erythrocytes (IEs): IEs are stained with SYBR Green I dye (DNA) to discriminate them from uninfected erythrocytes used as control. Supernatants are added on top of IEs and binding of specific antibodies is detected using a secondary-anti-human IgG (Fc-specific) antibody. Positive cultures were expanded and the VH and VL genes from individual clones were sequenced. Several antibodies showed a broad reactivity with the different isolates, while others were specific for a single isolate. The reactivity of the panel of antibodies isolated from donor C and donor D with erythrocytes infected with 8 different field isolates of P. falciparum (9106, 9605, 11019, 9215, 9775, 10975, 10936 and 11014) is shown below in Table 7. An example of IE staining is shown in FIG. 1.

[0486] Table 7 shows the panel of antibodies isolated from donor C and donor D (MGC1-MGD56; Table2) and their reactivity with erythrocytes infected with 8 different field isolates of P. falciparum (9106, 9605, 11019, 9215, 9775, 10975, 10936 and 11014). The numbers indicate the % of IEs that stained positive for the different antibodies. nd=not detectable.

TABLE-US-00019 % parasite recognition 9106 9605 11019 9215 9775 10975 10936 11014 Donor MGC1 6.7 19.5 32.7 14.9 5.7 1.4 2.0 3.4 C MGC2 5.6 22.4 11.9 28.8 2.9 2.6 2.8 1.5 MGC4 6.7 22.2 31.1 21.7 6.1 6.7 2.3 3.6 MGC5 6.6 20.3 37.6 26.4 6.0 3.8 2.1 2.9 MGC7 6.9 22.8 13.8 19.3 4.6 0.7 1.7 2.9 MGC17 1.3 6.8 7.1 16.7 2.5 1.5 2.4 1.6 MGC26 8.5 21.1 50.8 9.5 3.4 7.3 3.6 5.4 MGC28 7.5 20.9 30.0 10.8 9.8 12.3 3.0 2.8 MGC29 6.7 21.8 48.8 26.9 8.2 10.5 3.9 3.7 MGC32 7.8 22.9 38.1 13.1 7.9 3.2 2.9 4.5 MGC33 7.5 22.3 23.5 11.5 9.7 11.5 3.6 2.9 MGC34 6.8 23.7 34.1 27.1 17.5 15.2 11.3 11.4 MGC35 6.5 15.9 3.2 19.5 7.2 7.4 2.5 3.6 MGC36 6.9 17.9 17.9 12.4 6.2 8.6 2.7 4.6 MGC37 7.2 22.2 51.8 9.9 4.0 7.5 4.1 5.8 Donor MGD21 3.9 24.2 41.4 47.4 11.4 6.5 6.9 9.0 D MGD23 5.7 14.7 7.8 11.4 7.3 3.4 4.3 6.3 MGD30 4.2 7.4 4.4 9.0 5.6 6.5 2.6 3.4 MGD33 4.3 12.3 9.6 15.5 8.5 14.2 6.1 7.0 MGD34 5.0 28.4 46.6 35.7 16.0 11.2 8.1 13.0 MGD35 6.1 3.6 6.3 nd nd nd nd nd MGD39 13.7 31.7 43.0 37.4 15.0 14.1 10.5 11.5 MGD41 3.8 17.2 6.8 14.7 8.6 7.3 6.1 6.6 MGD47 10.7 28.7 24.6 22.3 14.4 11.2 11.3 10.2 MGD55 14.3 37.2 33.1 38.8 19.4 15.6 13.3 14.7 MGD56 3.3 17.3 4.3 12.0 6.6 6.7 2.4 9.5 <2% 2-5% 5-10% 10-20% 20-40% >40%

Example 2

The Human Monoclonal Antibodies that Broadly React with P. falciparum-Infected Erythrocytes are Characterized by a Large HCDR3 Containing a Mutated LAIR-1 Exon

[0487] The VH and VL sequences of all of the IE-specific human mAbs of Example 1 were aligned and the V, D and elements identified using the IMGT database. Surprisingly, all the broadly reactive mAbs isolated from both donors were characterized by an extraordinary long CDRH3 ranging from 120 to 130 amino acids, i.e. broadly reactive antibodies had an insert of more than 100 amino acids between the V and DJ segments, whereas narrowly reactive antibodies showed classical VD) organization of the heavy (H) chain gene. The middle and main part of this CDR3 was found to be highly homologous (92% to 98%) to the third exon plus a intronic sequence of LAIR1, a gene encoding an inhibitory receptor specific for collagen which is present on chromosome 19. The aminoacidic alignment of these unusual heavy chain variable regions (VH) is shown with reference to the genomic elements (exon and intron) of the LAIR1 gene (NCBI Reference Sequence: NC_018930.2) in FIG. 2 (cf. FIG. 2: alignment of the complete variable regions of selected antibodies to the genomic LAIR-1 portion corresponding to the inserts. LAIR-1 gene: ENSG00000167613). In addition, the LAIR-1 exon/intron insert was associated with VH4-4 and JH6 in donor D, and with VH3-7 and JH6 in donor C. All the antibodies carried several mutations both in the VD) elements and in the LAIR-1 insert. In both donors, the length and composition of VH and VL and the pattern of mutations define sister clones carrying different levels of mutations (Table 8).

[0488] Table 8 below shows the VH and VL gene usage of antibodies.

TABLE-US-00020 Heavy chain Light chain VH JH VL JL Donor MGC4 IGHV3-7 IGHJ6 IGLV7-43 IGLJ3 C MGC5 IGHV3-7 IGHJ6 IGLV7-43 IGLJ3 MGC8 1GHV3-7 IGHJ6 IGLV7-43 IGLJ3 MGC29 IGHV3-7 IGHJ6 IGLV7-43 IGLJ3 MGC33 IGHV3-7 IGHJ6 IGLV7-43 IGLJ3 MGC34 IGHV3-7 IGHJ6 IGLV7-43 IGLJ3 MGC35 IGHV3-7 IGHJ6 IGLV7-43 IGLJ3 MGC36 1GHV3-7 IGHJ6 IGLV7-43 IGLJ3 MGC2 IGHV3-7 IGHJ6 IGKV1-5 IGKJ2 MGC26 IGHV3-7 IGHJ6 IGKV1-5 IGKJ2 MGC37 IGHV3-7 IGHJ6 IGKV1-5 IGKJ2 MGC1 IGHV3-7 IGHJ6 IGKV4-1 IGKJ2 MGC17 IGHV3-7 IGHJ6 IGKV4-1 IGKJ2 MGC32 IGHV3-7 IGHJ6 IGKV4-1 IGKJ2 MGC7 IGHV3-7 IGHJ6 IGKV1-12 IGKJ4 Donor MGD21 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5 D MGD23 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5 MGD30 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5 MGD33 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5 MGD34 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5 MGD35 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5 MGD39 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5 MGD41 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5 MGD47 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5 MGD55 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5 MGD56 IGHV4-4 IGHJ6 IGKV1-8 IGKJ5

Example 3

Construction of Antibody Variants of MGD21

[0489] Of the antibodies described in Example 1 and Example 2 one broadly binding antibody, namely MGD21, was selected. MGD21 (SEQ ID NOs: 390-407) is a monoclonal antibody that binds to erythrocytes infected with 8/8 primary P. falciparum isolates and carries the LAIR-1 exon+intron insertion (a part of the intron, intron.sub., is shared with MGC antibodies, while the second part, intronp, is shared only with MGD antibodies). To understand which elements are required for binding to IEs, variants of the MGD21 mAb were produced, in which single elements (V, D, J and LAIR-1 exon and intron insertions) were either deleted or substituted with corresponding elements taken from an irrelevant antibody (F1499 reactive to influenza virus hemagglutinin, HA). In addition, variants were produced, in which somatic mutations were reverted to the germline configuration. In particular, mutations in the LAIR-1 exon+intron insertion were reverted to the corresponding original genomic sequence of LAIR-1 gene (NCBI Reference Sequence: NC_018930.2).

[0490] The following variants were produced, which are shown schematically in FIG. 3 (all the constructs have the same full complete constant region as antibody MGD21 as described herein and differ only in the heavy chain, while the light chain is not modified; the constructs are finally expressed as monoclonal antibodies (H+L chain)): [0491] 1. F1499V_DexinDJ is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable region of FI499 (VH1-69), the expression product of a first D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the mutated LAIR-1 fragment (Exon); the expression product of a LAIR-1 intron fragment (Intron); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of a J (Joining) gene segment element of a heavy chain variable region of MGD21 (JH6); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable region of MGD21 (VK1-8) and the expression product of a J (Joining) gene segment element of a light chain variable region of MGD21 (JK5); the expression product of a C (constant) gene segment of a light chain constant region. [0492] 2. FI1499VJ_DexinD is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable region of FI499 (VH1-69), the expression product of a first D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the mutated LAIR-1 fragment (Exon); the expression product of a LAIR-1 intron fragment (Intron); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of a J (Joining) gene segment element of a heavy chain variable region of FI499 (JH4); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable region of MGD21 (VK1-8) and the expression product of a J (Joining) gene segment element of a light chain variable region of MGD21 (JK5); the expression product of a C (constant) gene segment of a light chain constant region. [0493] 3. MGD21_exin_longGS is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable region of MGD21 (VH4-4); the expression product of a 10-amino-acid linker (GGGGS 2X); the mutated LAIR-1 fragment (Exon); the expression product of a LAIR-1 intron fragment (Introna); the expression product of a 20-amino-acid linker (GGGGS 4X); the expression product of a J (Joining) gene segment element of a heavy chain variable region of MGD21 (JH6);

[0494] the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable region of MGD21 (VK1-8) and the expression product of a J (Joining) gene segment element of a light chain variable region of MGD21 (JK5); the expression product of a C (constant) gene segment of a light chain constant region. [0495] 4. MGD21_exin_shortGS is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable region of MGD21 (VH4-4); the expression product of a 5-amino-acid linker (GGGGS 1 X); the mutated LAIR-1 fragment (Exon); the expression product of a LAIR-1 intron fragment (Introna); the expression product of a 5-amino-acid linker (GGGGS 1X); the expression product of a J (Joining) gene segment element of a heavy chain variable region of MGD21 (JH6); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable region of MGD21 (VK1-8) and the expression product of a J (Joining) gene segment element of a light chain variable region of MGD21 (JK5); the expression product of a C (constant) gene segment of a light chain constant region. [0496] 5. MGD21_NOexin is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable region of MGD21 (VH4-4); the expression product of a first D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of a J (Joining) gene segment element of a heavy chain variable region of MGD21 (JH6); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable region of MGD21 (VK1-8) and the expression product of a J (Joining) gene segment element of a light chain variable region of MGD21 (JK5); the expression product of a C (constant) gene segment of a light chain constant region. [0497] 6. MGD21_NOin is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable region of MGD21 (VH4-4); the expression product of a first D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the mutated LAIR-1 fragment (Exon); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of a J (Joining) gene segment element of a heavy chain variable region of MGD21 (JH6); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable region of MGD21 (VK1-8) and the expression product of a J (Joining) gene segment element of a light chain variable region of MGD21 (JK5); the expression product of a C (constant) gene segment of a light chain constant region. [0498] 7. MGD21_NOVD is formed by (in this order from N- to C-terminus): the mutated LAIR-1 fragment (Exon); the expression product of a LAIR-1 intron fragment (Intron); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of a J (Joining) gene segment element of a heavy chain variable region of MGD21 (JH6); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable region of MGD21 (VK1-8) and the expression product of a J (Joining) gene segment element of a light chain variable region of MGD21 (JK5); the expression product of a C (constant) gene segment of a light chain constant region.

[0499] 8. MGD21GL_exinWT is formed by (in this order from N- to C-terminus): the expression product of an unmutated V (variable) gene segment of a heavy chain variable region of MGD21 (VH4-4 GL); the expression product of a first D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the mutated LAIR-1 fragment (Exon); the expression product of a LAIR-1 intron fragment (Intron); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of an unmutated J (Joining) gene segment element of a heavy chain variable region of MGD21 (JH6 GL); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable region of MGD21 (VK1-8) and the expression product of a J (Joining) gene segment element of a light chain variable region of MGD21 (JK5); the expression product of a C (constant) gene segment of a light chain constant region. [0500] 9. MGD21_wholeGL is formed by (in this order from N- to C-terminus): the expression product of an unmutated V (variable) gene segment of a heavy chain variable region of MGD21 (VH4-4 GL); the expression product of a first D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the unmutated LAIR-1 fragment (Exon GL); the expression product of a unmutated LAIR-1 intron fragment (Intron GL); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of a J (Joining) gene segment element of a unmutated heavy chain variable region of MGD21 (JH6 GL); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of an unmutated V (variable) gene segment of a light chain variable region of MGD21 (VK1-8 GL) and the expression product of an unmutated J (Joining) gene segment element of a light chain variable region of MGD21 (JK5 GL); the expression product of a C (constant) gene segment of a light chain constant region. [0501] 10. MGD21 _irrelevant VK is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable region of MGD21 (VH4-4); the expression product of a first D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the mutated LAIR-1 fragment (Exon); the expression product of a LAIR-1 intron fragment (Intron); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of a J (Joining) gene segment element of a heavy chain variable region of MGD21 (1H6); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable region of FI499 (VK3-20) and the expression product of a J (Joining) gene segment element of a light chain variable region of F1499 (JK2); the expression product of a C (constant) gene segment of a light chain constant region. [0502] 11. MGD21_NOex is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable region of MGD21 (VH4-4); the expression product of a first D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of a LAIR-1 intron fragment (Intron); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of MGD21 (D.sub.); the expression product of a J (Joining) gene segment element of a heavy chain variable region of MGD21 (1H6); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable region of MGD21 (VK1-8) and the expression product of a J (Joining) gene segment element of a light chain variable region of MGD21 (JK5); the expression product of a C (constant) gene segment of a light chain constant region.

[0503] Table 9 below provides amino acid and nucleic acid sequences of the heavy chain variable regions of the constructs described above (Example 3).

TABLE-US-00021 TABLE 9 Sequences and Seq IDs of constructs SEQ ID NO Description Sequence* Heavy chain variable regions 594 FI499V_DexinDJ QVQPVQSGAEVKEPGSSVKVSCKTSGGLIRKSAVSWVRQAP aa GQGLEWMGGISALENTKDYAEKFQGRLTITADESTATAYMEL SSLTSEDTAIYYCATASPLKSQRDTDLPRPSISAEPGTVIPLGSHV TFVCRGPVGVQTFRLERERNYLYSDTEDVSQTSPSESEARFRID SVNAGNAGLFRCIYYKSRKWSEQSDYLELVVKGEDVTWALSQ SQDDPRACPQGELPISTDIYYVDVWGNGTTVTVSS 595 FI499V_DexinDJ CAGGTGCAGCCCGTCCAGTCTGGAGCAGAGGTGAAGGA nucl ACCTGGCAGCTCCGTGAAGGTCTCTTGCAAAACAAGTGG CGGGCTGATCCGCAAAAGTGCCGTGTCATGGGTCCGACA GGCTCCTGGACAGGGACTGGAATGGATGGGAGGCATCA GCGCACTGTTCAACACTAAGGACTACGCCGAAAAATTTCA GGGCCGGCTGACTATTACCGCCGATGAGAGTACAGCCAC TGCTTATATGGAACTGTCTAGTCTGACCAGCGAGGACACA GCTATCTACTATTGCGCAACCGCCTCACCACTGAAGTCCC AGAGAGACACCGACCTGCCAAGACCTTCCATCTCTGCAG AACCTGGCACAGTGATTCCACTGGGGTCCCACGTGACTTT CGTCTGTAGGGGACCAGTGGGCGTCCAGACCTTTCGCCT GGAGCGGGAAAGAAATTACCTGTATTCCGACACTGAGGA CGTGAGCCAGACCAGTCCCTCAGAGAGCGAAGCTAGGTT CCGCATCGATTCCGTGAACGCTGGGAATGCAGGACTGTT TAGATGCATCTACTATAAGTCTAGGAAATGGAGCGAGCA GTCCGACTACCTGGAACTGGTGGTCAAAGGGGAGGATG TGACATGGGCTCTGTCCCAGTCTCAGGACGATCCAAGAG CATGTCCCCAGGGCGAGCTGCCCATCTCTACTGACATCTA CTATGTGGATGTCTGGGGCAACGGGACCACAGTGACCGT CTCAAGC 596 FI499VJ_DexinD QVQPVQSGAEVKEPGSSVKVSCKTSGGLIRKSAVSWVRQAP aa GQGLEWMGGISALFNTKDYAEKFQGRLTITADESTATAYMEL SSLTSEDTAIYYCATASPLKSQRDTDLPRPSISAEPGTVIPLGSHV TFVCRGPVGVQTFRLERERNYLYSDTEDVSQTSPSESEARFRID SVNAGNAGLFRCIYYKSRKWSEQSDYLELVVKGEDVTWALSQ SQDDPRACPQGELPISTDIFDYWGQGTLVTVSS 597 FI499VJ_DexinD CAGGTGCAGCCCGTCCAGTCTGGAGCAGAGGTGAAGGA nucl ACCTGGCAGCTCCGTGAAGGTCTCTTGCAAAACAAGTGG CGGGCTGATCCGCAAAAGTGCCGTGTCATGGGTCCGACA GGCTCCTGGACAGGGACTGGAATGGATGGGAGGCATCA GCGCACTGTTCAACACTAAGGACTACGCCGAAAAATTTCA GGGCCGGCTGACCATTACAGCCGATGAGAGTACTGCCAC CGCTTATATGGAACTGTCTAGTCTGACCAGCGAGGACAC AGCTATCTACTATTGCGCAACCGCCTCACCACTGAAGTCC CAGAGAGACACCGACCTGCCAAGACCTTCCATCTCTGCA GAACCTGGCACAGTGATTCCACTGGGGTCCCACGTGACT TTCGTCTGTAGGGGACCAGTGGGCGTCCAGACCTTTCGC CTGGAGCGGGAAAGAAATTACCTGTATTCCGACACTGAG GACGTGAGCCAGACCAGTCCCTCAGAGAGCGAAGCTAG GTTCCGCATCGATTCCGTGAACGCTGGGAATGCAGGACT GTTTAGATGCATCTACTATAAGTCTAGGAAATGGAGCGAG CAGTCCGACTACCTGGAACTGGTGGTCAAAGGGGAGGA TGTGACTTGGGCTCTGTCCCAGTCTCAGGACGATCCAAG AGCATGTCCCCAGGGCGAGCTGCCCATCTCTACCGACAT TTTCGATTATTGGGGCCAGGGGACACTGGTGACTGTCTC AAGC 598 MGD21_exin_lo EVQLVETGPGLMKTSGTLSLTCAVSGDYVNTNRRWSWVRQ ngGS aa APGKGLEWIGEVHQSGRTNYNPSLKSRVTISVDKSKNQFSLKV DSVTAADTAVYYCARGGGGS GGGGSDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLER ERNYLYSDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCIYYKS RKWSEQSDYLELVVKGEDVTWALGGGGS GGGGS GGGGS GGGGSYYVDVWGNGTTVTVSS 599 MGD21_exin_lo GAAGTGCAGCTGGTGGAAACCGGCCCTGGACTGATGAA ngGS nucl GACCTCTGGCACACTGAGTCTGACATGCGCTGTGAGTGG GGACTACGTCAACACTAATCGGAGATGGTCTTGGGTGCG ACAGGCACCAGGAAAAGGACTGGAGTGGATCGGGGAA GTGCACCAGAGCGGAAGGACCAACTATAATCCTAGCCTG AAGTCCCGCGTGACAATTTCAGTCGATAAGAGCAAAAAC CAGTTCTCCCTGAAAGTGGACTCTGTCACTGCCGCTGATA CCGCAGTGTACTATTGTGCCAGAGGCGGGGGAGGCTCT GGGGGAGGCGGGAGTGACCTGCCCAGGCCTAGCATCTC CGCTGAACCAGGGACTGTGATTCCCCTGGGATCTCACGT GACCTTCGTCTGCAGAGGCCCTGTGGGGGTCCAGACATT TCGCCTGGAGCGGGAAAGAAACTACCTGTATTCTGACAC CGAGGATGTGAGTCAGACATCTCCCAGTGAGTCAGAAGC AAGGTTCCGCATCGATTCCGTCAACGCCGGAAATGCTGG CCTGTTTCGATGTATCTACTATAAGAGCCGGAAATGGAGC GAGCAGTCCGACTACCTGGAACTGGTGGTCAAGGGCGA GGATGTGACCTGGGCCCTGGGCGGGGGAGGCTCTGGG GGAGGCGGGAGTGGAGGCGGGGGATCAGGTGGAGGC GGGTCGTACTATGTGGACGTGTGGGGCAACGGGACCAC AGTGACCGTCAGCTCC 600 MGD21_exin_short EVQLVETGPGLMKTSGTLSLTCAVSGDYVNTNRRWSWVRQ GS aa APGKGLEWIGEVHQSGRTNYNPSLKSRVTISVDKSKNQFSLKV DSVTAADTAVYYCARGGGGS DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERERNYLY SDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCIYYKSRKWSE QSDYLELVVKGEDVTWALGGGGS YYVDVWGNGTTVTVSS 601 MGD21_exin_short GAAGTGCAGCTGGTGGAAACCGGCCCTGGACTGATGAA GS nucl GACCTCTGGCACACTGAGTCTGACATGCGCTGTGAGTGG GGACTACGTCAACACTAATCGGAGATGGTCTTGGGTGCG ACAGGCACCAGGAAAAGGACTGGAGTGGATCGGGGAA GTGCACCAGAGCGGAAGGACCAACTATAATCCTAGCCTG AAGTCCCGCGTGACAATTTCAGTCGATAAGAGCAAAAAC CAGTTCTCCCTGAAAGTGGACTCTGTCACTGCCGCTGATA CCGCAGTGTACTATTGTGCCAGAGGGGGAGGCGGGAGT GACCTGCCCAGGCCTAGCATCTCCGCTGAACCAGGGACT GTGATTCCCCTGGGATCTCACGTGACCTTCGTCTGCAGAG GCCCTGTGGGGGTCCAGACATTTCGCCTGGAGCGGGAA AGAAACTACCTGTATTCTGACACCGAGGATGTGAGTCAG ACATCTCCCAGTGAGTCAGAAGCAAGGTTCCGCATCGATT CCGTCAACGCCGGAAATGCTGGCCTGTTTCGATGTATCTA CTATAAGAGCCGGAAATGGAGCGAGCAGTCCGACTACCT GGAACTGGTGGTCAAGGGCGAGGATGTGACCTGGGCCC TGGGAGGCGGGGGATCATACTATGTGGACGTGTGGGGC AACGGGACCACAGTGACCGTCAGCTCC 602 MGD21_NOexin EVQLVETGPGLMKTSGTLSLTCAVSGDYVNTNRRWSWVRQ aa APGKGLEWIGEVHQSGRTNYNPSLKSRVTISVDKSKNQFSLKV DSVTAADTAVYYCARASPLKSQRDTGELPISTDIYYVDVWGN GTTVTVSS 603 MGD21_NOexin GAAGTGCAGCTGGTGGAAACCGGCCCTGGACTGATGAA nucl GACTTCAGGAACCCTGAGCCTGACTTGTGCCGTGAGCGG CGACTACGTCAACACCAATCGGAGATGGAGTTGGGTGCG GCAGGCACCAGGAAAAGGCCTGGAGTGGATCGGCGAA GTGCACCAGTCTGGGCGAACAAACTATAATCCCTCTCTGA AGAGTAGAGTGACTATTTCCGTGGACAAGTCTAAAAACCA GTTCAGCCTGAAAGTGGACTCCGTCACAGCCGCTGATAC TGCCGTGTACTATTGTGCAAGGGCCAGTCCCCTGAAGTC ACAGCGCGATACCGGGGAGCTGCCTATCAGCACAGACAT CTACTATGTGGATGTCTGGGGGAATGGAACCACAGTGAC AGTCAGCTCC 604 MGD21_NOin EVQLVETGPGLMKTSGTLSLTCAVSGDYVNTNRRWSWVRQ aa APGKGLEWIGEVHQSGRTNYNPSLKSRVTISVDKSKNQFSLKV DSVTAADTAVYYCARASPLKSQRDTDLPRPSISAEPGTVIPLGS HVTFVCRGPVGVQTFRLERERNYLYSDTEDVSQTSPSESEARF RIDSVNAGNAGLFRCIYYKSRKWSEQSDYLELVVKGELPISTDI YYVDVWGNGTTVTVSS 605 MGD21_NOin GAGGTGCAGCTGGTCGAAACCGGCCCAGGGCTGATGAA nucl GACTTCCGGAACCCTGTCTCTGACATGCGCCGTGTCCGG GGACTACGTCAACACTAATCGGAGATGGTCTTGGGTGAG GCAGGCTCCTGGAAAAGGCCTGGAGTGGATCGGGGAAG TGCACCAGTCCGGACGGACCAACTATAATCCATCTCTGAA GAGTAGAGTGACAATTAGTGTCGATAAGTCAAAAAACCA GTTCTCTCTGAAAGTGGACAGTGTCACAGCCGCTGATACT GCAGTGTACTATTGTGCAAGAGCAAGCCCCCTGAAGTCC CAGAGAGACACCGACCTGCCCAGGCCTTCTATCAGTGCT GAACCAGGCACTGTGATTCCCCTGGGGTCTCATGTGACC TTCGTCTGTAGAGGCCCCGTGGGAGTCCAGACTTTTCGC CTGGAGAGGGAACGCAATTACCTGTATTCAGACACCGAG GATGTGAGCCAGACATCACCTAGCGAGTCCGAAGCCCGA TTCCGGATCGACAGTGTGAACGCTGGAAATGCAGGCCTG TTTCGCTGTATCTACTATAAGAGCCGAAAATGGTCAGAGC AGAGCGATTACCTGGAACTGGTGGTCAAAGGCGAGCTG CCTATCAGCACTGACATCTACTATGTGGATGTCTGGGGGA ACGGAACCACAGTGACCGTCAGCTCC 606 MGD21_NOVD DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERERNYLY aa SDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCIYYKSRKWSE QSDYLELVVKGEDVTWALSQSQDDPRACPQGELPISTDIYYV DVWGNGTTVTVSS 607 MGD21_NOVD GACCTGCCACGACCATCTATTTCCGCCGAACCTGGGACT nucl GTCATTCCTCTGGGGAGCCACGTCACATTTGTCTGCCGG GGACCTGTCGGGGTGCAGACTTTCCGGCTGGAGCGGGA AAGAAACTACCTGTATTCTGACACCGAAGATGTGAGTCAG ACAAGCCCATCCGAGTCTGAAGCTAGGTTCCGCATCGAC TCCGTCAACGCCGGCAATGCTGGGCTGTTTCGATGCATCT ACTATAAGAGCAGAAAATGGAGCGAGCAGTCCGACTACC TGGAACTGGTGGTCAAGGGAGAGGATGTCACCTGGGCA CTGAGTCAGTCACAGGACGATCCCCGGGCCTGTCCTCAG GGCGAGCTGCCCATCAGCACTGATATCTACTATGTGGAT GTCTGGGGGAATGGCACTACTGTGACCGTCTCAAGC 608 MGD21GL_exin QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQP WT aa PGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSS VTAADTAVYYCARASPLKSQRDTDLPRPSISAEPGTVIPLGSHV TFVCRGPVGVQTFRLERERNYLYSDTEDVSQTSPSESEARFRID SVNAGNAGLFRCIYYKSRKWSEQSDYLELVVKGEDVTWALSQ SQDDPRACPQGELPISTDIYYMDVWGKGTTVTVSS 609 MGD21GL_exin CAGGTCCAGCTGCAGGAAAGCGGCCCAGGACTGGTGAA WT nucl GCCTAGCGGAACACTGAGTCTGACTTGTGCCGTGAGCGG AGGGAGCATCAGCTCCTCTAACTGGTGGTCTTGGGTGAG GCAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCGAAA TCTACCACAGCGGGTCCACCAACTATAATCCTTCACTGAA GAGCCGCGTGACAATCAGTGTGGACAAGTCAAAAAATCA GTTCAGCCTGAAACTGAGTTCAGTGACCGCCGCTGATAC AGCAGTCTACTATTGCGCACGGGCCAGCCCACTGAAATC CCAGCGAGACACTGATCTGCCACGGCCCTCTATCAGTGC TGAACCCGGAACAGTGATTCCTCTGGGCTCCCATGTGACT TTCGTCTGTCGCGGACCAGTGGGCGTCCAGACCTTTCGA CTGGAGCGGGAAAGAAACTACCTGTATTCTGACACTGAG GATGTGAGTCAGACCTCACCCAGCGAGTCCGAAGCCAG GTTCCGCATCGACAGCGTCAACGCTGGGAATGCAGGACT GTTTAGATGCATCTACTATAAGTCCAGGAAATGGTCCGAG CAGTCTGACTACCTGGAACTGGTGGTCAAGGGGGAGGA TGTGACATGGGCCCTGTCTCAGAGTCAGGACGATCCTAG AGCTTGTCCACAGGGCGAGCTGCCCATTTCAACCGATATC TATTACATGGATGTCTGGGGCAAGGGCACCACCGTGACC GTGAGCAGC 610 MGD21_wholeGL QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQP aa PGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSS VTAADTAVYYCARASPLKSQRDTEDLPRPSISAEPGTVIPLGSH VTFVCRGPVGVQTERLERESRSTYNDTEDVSQASPSESEARFRI DSVSEGNAGPYRCIYYKPPKWSEQSDYLELLVKGEDVTWALP QSQLDPRACPQGELPISTDIYYMDVWGKGTTVTVSS 611 MGD21_wholeGL CAGGTGCAGCTGCAGGAAAGCGGACCAGGCCTGGTCAA nucl GCCCTCAGGCACTCTGAGCCTGACCTGCGCTGTGAGTGG CGGGTCAATCAGCTCCTCTAATTGGTGGTCCTGGGTGAG GCAGCCCCCTGGGAAAGGACTGGAGTGGATCGGCGAAA TCTACCACTCTGGGAGTACAAACTATAATCCCAGCCTGAA GTCCCGCGTGACTATTTCCGTGGACAAGTCTAAAAATCAG TTCAGCCTGAAACTGAGTTCAGTGACAGCCGCTGATACTG CAGTCTACTATTGCGCACGAGCCAGTCCTCTGAAGTCCCA GCGGGACACTGAGGACCTGCCTAGACCATCAATCAGCGC CGAGCCTGGAACTGTGATTCCACTGGGCTCTCATGTGAC CTTCGTCTGTAGAGGACCAGTGGGAGTCCAGACCTTCCG GCTGGAGAGAGAATCCCGATCTACCTACAACGACACAGA AGATGTGAGCCAGGCTAGTCCATCAGAGAGCGAAGCAC GGTTTAGAATCGACTCCGTGTCTGAGGGGAATGCCGGAC CCTACAGATGCATCTACTATAAGCCACCCAAATGGTCTGA GCAGAGTGACTATCTGGAACTGCTGGTGAAAGGAGAGG ATGTCACCTGGGCACTGCCTCAGTCTCAGCTGGACCCCA GAGCTTGTCCTCAGGGAGAGCTGCCTATCAGCACCGACA TCTACTATATGGACGTGTGGGGCAAAGGGACCACAGTGA CAGTCAGCTCCGCGTCGACTTCGCA 612 MGD21_NOex EVQLVETGPGLMKTSGTLSLTCAVSGDYVNTNRRWSWVRQ aa APGKGLEWIGEVHQSGRTNYNPSLKSRVTISVDKSKNQFSLKV DSVTAADTAVYYCARASPLKSQRDTGEDVTWALSQSQDDPR ACPQGELPISTDIYYVDVWGNGTTVTVSS 613 MGD21_NOex GAAGTGCAGCTGGTGGAAACCGGCCCTGGACTGATGAA nucl GACTTCCGGAACCCTGTCTCTGACTTGCGCCGTGTCTGGC GACTACGTCAACACCAATCGGAGATGGAGCTGGGTGCG GCAGGCTCCAGGAAAAGGCCTGGAGTGGATCGGCGAAG TGCACCAGTCCGGGCGAACAAACTATAATCCCTCACTGAA GAGCAGAGTGACTATTAGTGTCGATAAGTCAAAAAACCA GTTCTCTCTGAAAGTGGACAGTGTCACAGCCGCTGATACT GCCGTGTACTATTGCGCAAGGGCCAGCCCTCTGAAGTCC CAGAGAGACACCGGGGAGGATGTGACATGGGCTCTGTC TCAGAGTCAGGACGATCCCCGGGCATGTCCTCAGGGCG AACTGCCAATCAGCACCGACATCTACTATGTGGATGTCTG GGGGAATGGAACCACAGTGACAGTCAGCTCC

Example 4

Identification of the Mutated LAIR-1 Exon as the Only Element Required for MGD21 mAb Binding to P. falciparum-Infected Erythrocytes (IEs)

[0504] The 10 antibody variants constructed in Example 3 as well as the antibody MGD21 (cf. Examples 1 and 2) and the antibody FI499 (control: irrelevant antibody reactive to influenza virus hemagglutinin, HA) were expressed in HEK 293 cells and tested for their capacity to stain IEs as described in Example 1. Briefly, IEs are stained with SYBR Green I dye (DNA) to discriminate them from uninfected erythrocytes used as control. The antibody variants are added on top of IEs and binding of specific antibodies to IEs is detected using a secondary-anti-human IgG (Fc -specific) antibody. The binding data are shown in FIG. 4. Most constructs show binding to IEs, with the exception of those constructus wherein the exon is either not present or is in the original genomic form (Con5/MGD21_NOexin, Con9/MGD21_wholeGL and Con11 MGD21_NOex). The results indicate that the only element required for binding to IEs is the mutated LAIR-1 exon.

Example 5

Construction of Ig Fusion Proteins Comprising the Mutated LAIR-1 Fragment

[0505] To investigate whether the mutated LAIR-1 exon alone is sufficient to bind to IEs, six different Ig fusion proteins comprising the mutated LAIR-1 fragment were constructed by inserting: [0506] (a) the mutated LAIR-1 exon, preferably according to SEQ ID NO: 84 or a functional sequence variant thereof; [0507] (b) optionally, one or more further elements (intron segments) of LAIR-1, preferably corresponding to such elements of the antibody MGD21 as shown in FIG. 5 and; [0508] (c) optionally, one or more different elements of a heavy chain variable region of an IgG-type antibody, preferably of the antibody MGD21,

[0509] into a plasmid designed for expression of mouse IgG2b fusion proteins (pINFUSE-rnIgG2b-Fc2 by Invivogen) or human IgG1 fusion proteins (pINFUSE-hIgG1-Fc2 by Invivogen). Preferred sequences for the constant regions (hinge region and CH2 and CH3 domains) of mouse IgG2b fusion proteins comprise or consist of a sequence according to SEQ ID NO: 614 (amino acid) or SEQ ID NO: 615 (nucleic acid), or functional sequence variants thereof. Preferred sequences for the constant regions (hinge region and CH2 and CH3 domains) of human IgG1 fusion proteins comprise or consist of a sequence according to SEQ ID NO: 616 (amino acid) or SEQ ID NO: 617 (nucleic acid), or functional sequence variants thereof. Preferably, the mutated LAIR-1 fragment (Exon) in the following Ig fusion proteins comprises or consists of an amino acid sequence according to SEQ ID NO: 83 or a functional sequence variant thereof.

[0510] The different fusion proteins are shown schematically in FIG. 5 in comparison to the antibody MGD21 and described in the following: [0511] 1. M1 (also referred to as DexinDJ-mIgG2b) is formed by (in this order from N- to C-terminus): the expression product of a first D (Diversity) gene segment element of a heavy chain variable region of an IgG-type antibody, preferably of MGD21 (D.sub.); the mutated LAIR-1 fragment (Exon); the expression product of a LAIR-1 intron fragment (Intron); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of an IgG-type antibody, preferably of MGD21 (Dr); the expression product of a J (Joining) gene segment element of a heavy chain variable region of an IgG-type antibody, preferably of MGD21 (JH6); followed by a hinge region and CH2 and CH3 domains from mouse IgG2b. [0512] An exemplary variable region of such an M1 fusion protein, which is particularly preferred, comprises or consists of an amino acid sequence according to SEQ ID NO: 618 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 619 or by a functional sequence variant thereof. More preferably, a complete M1 fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 620 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 621 or by a functional sequence variant thereof. [0513] 2. M2 (also referred to as exinDJ-mIgG2b) is formed by (in this order from N- to C-terminus): the mutated LAIR-1 fragment (Exon); the expression product of a LAIR-1 intron fragment (Intron); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of an IgG-type antibody, preferably of MGD21 (D.sub.); the expression product of a J (Joining) gene segment element of a heavy chain variable region of an IgG-type antibody, preferably of MGD21 (JH6); followed by a hinge region and CH2 and CH3 domains from mouse IgG2b. [0514] An exemplary variable region of such an M2 fusion protein, which is particularly preferred, comprises or consists of an amino acid sequence according to SEQ ID NO: 624 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 625 or by a functional sequence variant thereof. More preferably, a complete M2 fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 626 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 627 or by a functional sequence variant thereof. [0515] 3. M3 (also referred to as exin-mIgG2b) is formed by (in this order from N- to C-terminus): the mutated LAIR-1 fragment (Exon); the expression product of a partial LAIR-1 intron fragment (Intron.sub.); followed by a hinge region and CH2 and CH3 domains from mouse IgG2b. [0516] An exemplary variable region of such an M3 fusion protein, which is particularly preferred, comprises or consists of an amino acid sequence according to SEQ ID NO: 628 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 629 or by a functional sequence variant thereof. More preferably, a complete M3 fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 630 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 631 or by a functional sequence variant thereof. [0517] 4. M4 (also referred to as ex-mIgG2b) is formed by (in this order from N- to C-terminus): the mutated LAIR-1 fragment (Exon); followed by a hinge region and CH2 and CH3 domains from mouse IgG2b. [0518] An exemplary variable region of such an M4 fusion protein, which is particularly preferred, comprises or consists of an amino acid sequence according to SEQ ID NO: 632 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 633 or by a functional sequence variant thereof. More preferably, a complete M4 fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 634 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 635 or by a functional sequence variant thereof. [0519] 5. H1 (also referred to as DexinDJ-hIgG1) is formed by (in this order from N- to C-terminus): the expression product of a first D (Diversity) gene segment element of a heavy chain variable region of an IgG-type antibody, preferably of MGD21 (D.sub.); the mutated LAIR-1 fragment (Exon); the expression product of a LAIR-1 intron fragment (Intron); the expression product of a second D (Diversity) gene segment element of a heavy chain variable region of an IgG-type antibody, preferably of MGD21 (D.sub.); the expression product of a J (Joining) gene segment element of a heavy chain variable region of an IgG-type antibody, preferably of MGD21 (JH6); followed by a hinge region and CH2 and CH3 domains from human IgG1. [0520] An exemplary variable region of such an H1 fusion protein, which is particularly preferred, comprises or consists of an amino acid sequence according to SEQ ID NO: 618 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 619 or by a functional sequence variant thereof. More preferably, a complete H1 fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 622 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 623 or by a functional sequence variant thereof. [0521] 6. H2 (also referred to as ex-hIgG1) is formed by (in this order from N- to C-terminus): the mutated LAIR-1 fragment (Exon); followed by a hinge region and CH2 and CH3 domains from human IgG1. [0522] An exemplary variable region of such an H2 fusion protein, which is particularly preferred, comprises or consists of an amino acid sequence according to SEQ ID NO: 632 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 633 or by a functional sequence variant thereof. More preferably, a complete H2 fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 636 or according to a functional sequence variant thereof, which may preferably be encoded by a nucleic acid sequence according to SEQ ID NO: 637 or by a functional sequence variant thereof.

[0523] Table 10 below shows the amino acid and nucleotide sequences of the antibody constructs of Example 5, whereby the constant chain sequences are identical for the mouse IgG2b-antibody constructs M1, M2, M3, and M4 (mIgG2b) and for the human IgG1-antibody constructs H1 and H2 (hIgG1).

TABLE-US-00022 TABLE 10 Sequences and Seq IDs of Ig fusion proteins SEQ ID NO Description Sequence* Constant chains 614 mIgG2b aa AMVRSPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKD VLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQ THREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIE RTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDIS VEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKT DSFSCNVRHEGLKNYYLKKTISRSPGK 615 mIgG2b nucl GCCATGGTTAGATCTCCCAGCGGGCCCATTTCAACAATCA ACCCCTGTCCTCCATGCAAGGAGTGTCACAAATGCCCAG CTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCC TCCAAATATCAAGGATGTACTCATGATCTCCCTGACACCC AAGGTCACGTGTGTGGTGGTGGATGTGAGCGAGGATGA CCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGA AGTACACACAGCTCAGACACAAACCCATAGAGAGGATTA CAACAGTACTATCCGGGTGGTCAGCACCCTCCCCATCCA GCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCA AGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAA CCATCTCAAAAATTAAAGGGCTAGTCAGAGCTCCACAAGT ATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAA AGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCT GGAGACATCAGTGTGGAGTGGACCAGCAATGGGCATAC AGAGGAGAACTACAAGGACACCGCACCAGTCCTGGACTC TGACGGTTCTTACTTCATATATAGCAAGCTCAATATGAAAA CAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAACG TGAGACACGAGGGTCTGAAAAATTACTACCTGAAGAAGA CCATCTCCCGGTCTCCGGGTAAA 616 hIgG1 aa AMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 617 hIgG1 nucl GCCATGGTTAGATCTGACAAAACTCACACATGCCCACCGT GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC ACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA MGD21-DexinDJ-mIgG2b 618 DexinDJ variable ASPLKSQRDTDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTF part aa RLERERNYLYSDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCI YYKSRKWSEQSDYLELVVKGEDVTWALSQSQDDPRACPQGE LPISTDIYYVDVWGNGTTVTVSS 619 DexinDJ variable gcgtctccactcaaatctcagagggacaccgatctgcccagaccctccatctcggctg part nucl agccgggcaccgtgatccccctggggagccatgtgactacgtgtgccggggcccggt tggggttcaaacattccgcctggagagggagaggaattatttatacagtgatactgaaga tgtgtctcaaactagtccatctgagtcggaggccagattccgcattgactcagtaaatgc aggcaatgccgggctttttcgctgcatctattacaagtcccgtaaatggtctgagcagag tgactacctggagctggtggtgaaaggtgaggacgtcacctgggccctgtcccagtctc aagacgaccctcgagcttgtccccagggggagctccccataagtaccgatatttacta cgtggacgtctggggcaacgggaccacggtcaccgtctcctca 620 DexinDJ-mIgG2b ASPLKSQRDTDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTF complete RLERERNYLYSDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCI sequence aa YYKSRKWSEQSDYLELVVKGEDVTWALSQSQDDPRACPQGE LPISTDIYYVDVWGNGTTVTVSSAMVRSPSGPISTINPCPPCKE CHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSED DPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQH QDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPP AEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAP VLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKK TISRSPGK 621 DexinDJ-mIgG2b gcgtctccactcaaatctcagagggacaccgatctgcccagaccctccatctcggctg complete agccgggcaccgtgatccccctggggagccatgtgactttcgtgtgccggggcccggt sequence nucl tggggttcaaacattccgcctggagagggagaggaattatttatacagtgatactgaaga tgtgtctcaaactagtccatctgagtcggaggccagattccgcattgactcagtaaatgc aggcaatgccgggctttttcgctgcatctattacaagtcccgtaaatggtctgagcagag tgactacctggagctggtggtgaaaggtgaggacgtcacctgggccctgtcccagtctc aagacgaccctcgagcttgtccccagggggagctccccataagtaccgatatttacta cgtggacgtctggggcaacgggaccacggtcaccgtctcctcaGCCATGGTTA GATCTCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCC TCCATGCAAGGAGTGTCACAAATGCCCAGCTCCTAACCTC GAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCA AGGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTG TGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCC AGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAG CTCAGACACAAACCCATAGAGAGGATTACAACAGTACTAT CCGGGTGGTCAGCACCCTCCCCATCCAGCACCAGGACTG GATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAA AGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAATT AAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCG CCACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTC ACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGT GTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTA CAAGGACACCGCACCAGTCCTGGACTCTGACGGTTCTTA CTTCATATATAGCAAGCTCAATATGAAAACAAGCAAGTGG GAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAG GGTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGT CTCCGGGTAAA 622 DexinDJ-hIgG1 ASPLKSQRDTDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTF complete RLERERNYLYSDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCI sequence aa YYKSRKWSEQSDYLELVVKGEDVTWALSQSQDDPRACPQGE LPISTDIYYVDVWGNGTTVTVSSAMVRSDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLICLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFEL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 623 DexinDJ-hIgG1 gcgtctccactcaaatctcagagggacaccgatctgcccagaccctccatctcggctg complete agccgggcaccgtgatccccctggggagccatgtgactttcgtgtgccggggcccggt sequence nucl tggggttcaaacattccgcctggagagggagaggaattattlatacagtgatactgaaga tgtgtctcaaactagtccatctgagtcggaggccagattccgcattgactcagtaaatgc aggcaatgccgggctttttcgctgcatctattacaagtcccgtaaatggtctgagcagag tgactacctggagctggtggtgaaaggtgaggacgtcacctgggccctgtcccagtctc aagacgaccctcgagcttgtccccagggggagctccccataagtaccgatatttacta cgtggacgtctggggcaacgggaccacggtcaccgtctcctcaGCCATGGTTA GATCTGACAAAACTCACACATGCCCACCGTGCCCAGCAC CTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG GTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC TCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA AGAGCCTCTCCCTGTCTCCGGGTAAA MGD21-exinDJ-mIgG2b 624 exinDJ variable DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERERNYLY part aa SDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCIYYKSRKWSE QSDYLELVVKGEDVTWALSQSQDDPRACPQGELPISTDIYYV DVWGNGTTVTVSS 625 exinDJ variable gatctgcccagaccctccatctcggctgagccgggcaccgtgatccccctggggagc part nucl catgtgactttcgtgtgccggggcccggttggggttcaaacattccgcctggagaggga gaggaattatttatacagtgatactgaagatgtgtctcaaactagtccatctgagtcggag gccagattccgcattgactcagtaaatgcaggcaatgccgggctttttcgctgcatctatt acaagtcccgtaaatggtctgagcagagtgactacctggagctggtggtgaaaggtga ggacgtcacctgggccctgtcccagtctcaagacgaccctcgagcttgtccccaggg ggagctccccataagtaccgatatttactacgtggacgtctggggcaacgggaccacg gtcaccgtctcctca 626 exinDJ-mIgG2b DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERERNYLY complete SDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCIYYKSRKWSE sequence aa QSDYLELVVKGEDVTWALSQSQDDPRACPQGELPISTDIYYV DVWGNGTTVTVSSAMVRSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 627 exinDJ-mIgG2b gatctgcccagaccctccatctcggctgagccgggcaccgtgatccccctggggagc complete catgtgactttcgtgtgccggggcccggttggggttcaaacattccgcctggagaggga sequence nucl gaggaattatttatacagtgatactgaagatgtgtctcaaactagtccatctgagtcggag gccagattccgcattgactcagtaaatgcaggcaatgccgggctttttcgctgcatctatt acaagtcccgtaaatggtctgagcagagtgactacctggagctggtggtgaaaggtga ggacgtcacctgggccctgtcccagtctcaagacgaccctcgagcttgtccccaggg ggagctccccataagtaccgatatttactacgtggacgtctggggcaacgggaccacg gtcaccgtctcctcaGCCATGGTTAGATCTGACAAAACTCACACA TGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCC CAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCC GGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGC CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA MGD21-exin-mIgG2b 628 exin variable part DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTERLERERNYLY aa SDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCIYYKSRKWSE QSDYLELVVKGEDVTWAL 629 exin variable part gatctgcccagaccctccatctcggctgagccgggcaccgtgatccccctggggagc nucl catgtgactttcgtgtgccggggcccggttggggttcaaacattccgcctggagaggga gaggaattatttatacagtgatactgaagatgtgtctcaaactagtccatctgagtcggag gccagattccgcattgactcagtaaatgcaggcaatgccgggctttttcgctgcatctatt acaagtcccgtaaatggtctgagcagagtgactacctggagctggtggtgaaaggtga ggacgtcacctgggccctg 630 exin-mIgG2b DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTERLERERNYLY complete SDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCIYYKSRKWSE sequence aa QSDYLELVVKGEDVTWALAMVRSDKTHTCPPCPAPELLGGPS VFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 631 exin-mIgG2b gatctgcccagaccctccatctcggctgagccgggcaccgtgatccccctggggagc complete catgtgactttcgtgtgccggggcccggttggggttcaaacattccgcctggagaggga sequence nucl gaggaattatttatacagtgatactgaagatgtgtctcaaactagtccatctgagtcggag gccagattccgcattgactcagtaaatgcaggcaatgccgggcatttttcgctgcatctatt acaagtcccgtaaatggtctgagcagagtgactacctggagctggtggtgaaaggtga ggacgtcacctgggccctgGCCATGGTTAGATCTGACAAAACTCA CACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCT GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAA MGD21-ex-mIgG2b 632 exon variable DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERERNYLYSDTEDVSQTS part aa PSESEARFRIDSVNAGNAGLFRCIYYKSRKWSEQSDYLELVVK 633 exon variable gatctgcccagaccctccatctcggctgagccgggcaccgtgatccccctggggagc part nucl catgtgactttcgtgtgccggggcccggttggggttcaaacattccgcctggagaggga gaggaattatttatacagtgatactgaagatgtgtctcaaactagtccatctgagtcggag gccagattccgcattgactcagtaaatgcaggcaatgccgggctttttcgctgcatctatt acaagtcccgtaaatggtctgagcagagtgactacctggagctggtggtgaaa 634 ex-mIgG2b DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERERNYLY complete SDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCIYYKSRKWSE sequence aa QSDYLELVVKAMVRSPSGPISTINPCPPCKECHKCPAPNLEGG PSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKC KVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLT CLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYS KLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK 635 ex-mIgG2b gatctgcccagaccctccatctcggctgagccgggcaccgtgatccccctggggagc complete catgtgactttcgtgtgccggggcccggttggggttcaaacattccgcctggagaggga sequence nucl gaggaattatttatacagtgatactgaagatgtgtctcaaactagtccatctgagtcggag gccagattccgcattgactcagtaaatgcaggcaatgccgggctttttcgctgcatctatt acaagtcccgtaaatggtctgagcagagtgactacctggagctggtggtgaaaGCC ATGGTTAGATCTCCCAGCGGGCCCATTTCAACAATCAACC CCTGTCCTCCATGCAAGGAGTGTCACAAATGCCCAGCTCC TAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCA AATATCAAGGATGTACTCATGATCTCCCTGACACCCAAGG TCACGTGTGTGGTGGTGGATGTGAGCGAGGATGACCCA GACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTA CACACAGCTCAGACACAAACCCATAGAGAGGATTACAAC AGATACTATCCGGGTGGTCAGCACCCTCCCCATCCAGCAC CAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGT CAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATC TCAAAAATTAAAGGGCTAGTCAGAGCTCCACAAGTATACA TCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAGATG TCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGA CATCAGTGTGGAGTGGACCAGCAATGGGCATACAGAGG AGAACTACAAGGACACCGCACCAGTCCTGGACTCTGACG GTTCTTACTTCATATATAGCAAGCTCAATATGAAAACAAGC AAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGA CACGAGGGTCTGAAAAATTACTACCTGAAGAAGACCATCT CCCGGTCTCCGGGTAAA 636 ex-hIgG1 DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERERNYLY complete SDTEDVSQTSPSESEARFRIDSVNAGNAGLFRCIYYKSRKWSE sequence aa QSDYLELVVKAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 637 ex-hIgG1 gatctgcccagaccctccatctcggctgagccgggcaccgtgatccccctggggagc complete catgtgactttcgtgtgccggggcccggttggggttcaaacattccgcctggagaggga sequence nucl gaggaattatttatacagtgatactgaagatgtgtctcaaactagtccatctgagtcggag gccagattccgcattgactcagtaaatgcaggcaatgccgggctttttcgctgcatctatt acaagtcccgtaaatggtctgagcagagtgactacctggagctggtggtgaaaGCC ATGGTTAGATCTGACAAAACTCACACATGCCCACCGTGCC CAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTT CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

Example 6

Ig Fusion Proteins Comprising the Mutated LAIR-1 Fragment Bind to IEs

[0524] The four exemplary mouse IgG2b fusion proteins constructed in Example 5 (i.e. one of each type: M1, M2, M3, and M4), which were consisting of amino acid sequences as outlined for the complete fusion protein, respectively, were used to investigate whether the mutated LAIR-1 fragment is sufficient to bind to infected erythrocytes (IEs). To this end, HEK 293 cells were transfected with the fusion proteins only and supernatants were collected and tested for binding to IEs as described in Example 1. Briefly, IEs are stained with SYBR Green I dye (DNA) to discriminate them from uninfected erythrocytes used as control. The surnatants are added on top of IEs and binding of fusion proteins to IEs is detected using a secondary-anti-human or anti-mouse IgG (Fc -specific) antibody.

[0525] All fusion proteins were found to bind to infected erythrocytes (FIG. 6). These results identify the mutated LAIR-1 fragment as a unique domain that binds to P. falciparum-infected erythrocytes.

Example 7

Antibodies and Ig Fusion Proteins Efficiently Opsonize and Agglutinate P. falciparum-Infected Erythrocytes

[0526] To investigate the potential therapeutic impact of selected broadly reactive antibodies of Example 1 and of the Ig fusion proteins constructed in Example 5, i.e. whether these antibodies/fusion proteins could opsonize infected erythrocytes and thus mediate their phagocytosis and destruction by mononuclear phagocytes, their capacity to opsonize infected erythrocytes was measured.

[0527] To this end, P. falciparum (3D7) were stained with DAPI and mixed with different concentrations of the two exemplary human IgG1 fusion proteins constructed in Example 5 (i.e. one of each type: H1 and H2), which were consisting of amino acid sequences as outlined for the complete fusion protein, respectively. Thereafter, they were incubated with human monocytes at 37 C. for 1 hour.

[0528] Thereafter, monocytes were stained with anti-CD14-APC to measure the fraction of monocytes that contained parasites. The results are shown in FIG. 7 with FIG. 7A showing the MFI (mean fluorecnce intensity) of DAPI and FIG. 7B showing the percentage of DAPI-positive monocytes calculated in CD14-positive populations.

[0529] The results demonstrate that low concentrations of the two exemplary human IgG1 fusion proteins constructed in Example 5 can efficiently opsonize infected erythrocytes. These findings indicate that the Ig fusion proteins constructed in Example 5 can potently mediate phagocytosis and destruction of infected erythrocytes in vivo.

[0530] Finally, it was tested whether the antibodies MGD21 and MGC34 were able to agglutinate erythrocytes infected with P. falciparum 3D7 or the Kenyan P. falciparum isolate 11019. As shown in FIG. 7C MGD21, as well as MGC34, could agglutinate erythrocytes infected with 3D7 or the Kenyan isolate 11019.

[0531] Next, P. falciparum (3D7 or 11019) were stained with DAPI and mixed with different concentrations of the five broadly reactive antibodies described in Table 2 and Example 1 (i.e. one of each type: MGD21, MGD47, MGD55, MGC28 and MGC34). BKC3 was used as control. Thereafter, they were incubated with human monocytes at 37 C. for 1 hour and, then, monocytes were stained with anti-CD14-APC to measure the fraction of monocytes that contained parasites. The results are shown in FIG. 8 with FIG. 8A showing the MFI (mean fluorecnce intensity) of DAPI in 3D7 and FIG. 8B showing the MFI (mean fluorecnce intensity) of DAPI in 11019-MGD21.sup.+IEs. Results show that low concentrations of all five antibodies tested constructed (MGD21, MGD47, MGD55, MGC28 and MGC34; cf. Table 2) can efficiently opsonize infected erythrocytes, whereas MGD21 LALA and BKC3 controls show no effect. These findings indicate that the broadly reactive antibodies can potently mediate phagocytosis and destruction of infected erythrocytes in vivo.

Example 8

A Model of the Mutated LAIR-1 Fragment: Somatic Mutations in the LAIR-1 Fragment are Critical Both for Binding IEs and Losing Binding to Collagen

[0532] The mutated LAIR-1 fragment of the antibodies of Example 1 has a sequence homology ranging from 84% to 96% with the amino acids 24 to 121 of native human LAIR-1 (SEQ ID NO: 14; for example: MGD53_exon=96%; MGC2_exon=91%; MGD21_exon=86%; MGD35_exon=84%). FIG. 9 shows an alignment of the mutated LAIR-1 exon of the human monoclonal antibodies of Example 1 (cf. SEQ ID NOs 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 and 103Table 1) with amino acids 24 to 121 of native human LAIR-1 (SEQ ID NO: 14).

[0533] From the human monoclonal antibodies of Example 1 those antibodies were selected, which most strongly bind to the most of the IEs infected with different P. falciparum strains (broadest binding to IEs). These were MGD21, MGD34, MGD39, MGD47, and MGD55 (cf. Table 7 of Example 1). An alignment of the amino acid sequences of the LAIR-1 exon fragment of these antibodies, i.e. amino acid sequences according to SEQ ID NOs: 83, 91, 95, 99 and 101 with an exemplary genomic LAIR-1 sequence, revealed five mutated residues, which are crucial to increase the affinity and the breadth of binding to P. falciparum-IEs. The same five mutated residues were also found to be important for losing binding to collagen that is the natural ligand of the native LAIR-1 receptor (see Example 9). The five crucial positions are T67, N69, A77, P106 and P107 and are shown in frames in FIG. 9.

[0534] The mutated LAIR-1 fragment according to the present invention was modelled based on a crystal structure of native LAIR-1 extracellular domain (residues: 24 to 121) (FIG. 10; for the crystal structure of native LAIR-1 see MMDB ID: 78950, PDB ID: 3KGR). According to the crystal structure of LAIR-1, at least one of the following five residues must be mutated to lose collagen binding and to gain binding to infected erythrocytes (positions are defined in respect to the amino acid sequence of native human LAIR-1):

[0535] T67, N69, A77, P106, and P107 (FIG. 10).

[0536] Preferred mutations are shown below in Table 11, with T67L, N69S, A77T, P106S, and P107R being the most preferred mutations for each of the five positions.

TABLE-US-00023 TABLE 11 preferred mutations for each of the five positions in the mutated LAIR-1 fragment. Position Mutation T67 T67L, T67G, T67I, T67R, T67K N69 N69S, N69T A77 A77T, A77P, A77V P106 P106S, P106A, P106D P107 P107R, P107S

Example 9

Identification of Mutations of LAIR1 Fragment that are Crucial for Binding to P. falciparum-IE

[0537] To identify which of the five mutations are crucial for binding to IEs, fusion proteins comprising the LAIR-1 fragment, which was either unmutated (SEQ ID NO: 14) or carrying one or more of the following five mutations: T67L (L); N69S (Si); A77T (T); P106S (S2); and P107R (R), were produced. The principal structure of these fusion proteins (i.e. except for the mutated LAIR-1 fragment) is identical to that of H2 of Example 5 as described above (also referred to as ex-hIgG1). While in the construct H2 of Example 5 (also referred to as ex-hIgG1) the mutated LAIR-1 exon of the antibody MGD21 was used (SEQ ID NO: 83), the present constructs are instead based on the native human LAIR-1 fragment (amino acids 24-121; SEQ ID NO: 14) and differ from that (i.e. from SEQ ID NO: 14) only in one or more of the following five mutations: T67L (L); N695 (S1); A77T (T); P106S (S2); and P107R (R).

[0538] Table 12 shows SEQ ID and sequences of the different fusion proteins.

TABLE-US-00024 TABLE 12 Sequences and Seq ID NOs of the LAIR-1 Ig fusion protein constructs of Example 9, whereby only the sequences of the (mutated) LAIR-1 fragment are shown. Mutations in comparison to native human LAIR-1 (SEQ ID NO: 14) are shown underlined in the amino acid sequence. SEQ ID NO Description Sequence* 14 LAIR1ex aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY NDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSE QSDYLELLVK 638 LAIR1ex nucl GAGGACCTGCCAAGACCCAGCATCTCCGCAGAACCTGG GACTGTGATTCCACTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTACCTACAACGACACAGAGGACGTGAG CCAGGCCTCACCCAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAATGCCGGCCCTTACAGATG CATCTACTATAAGCCCCCTAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 23 LAIR1ex +L aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSLY NDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSE QSDYLELLVK 24 LAIR1ex +L nucl GAGGACCTGCCAAGACCCAGCATCTCCGCAGAACCTGG GACCGTGATTCCACTGGGCTCCCACGTGACATTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACTTTTAGGCTGGAGCG CGAATCTCGAAGTCTGTACAACGACACAGAGGACGTGAG CCAGGCCTCACCAAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAATGCCGGCCCTTACAGATG CATCTACTATAAGCCCCCTAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 25 LAIR1ex +LR aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSLY NDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPRKWSE QSDYLELLVK 26 LAIR1ex +LR nucl GAGGACCTGCCCCGCCCTAGCATCTCCGCAGAACCAGG GACCGTGATTCCCCTGGGCTCCCACGTGACATTCGTCTGC AGGGGCCCCGTGGGAGTCCAGACTTTTAGGCTGGAGCG CGAATCTCGAAGTCTGTACAACGACACCGAGGACGTGAG CCAGGCCTCACCTAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAATGCCGGCCCTTACAGATG CATCTACTATAAGCCAAGAAAATGGTCAGAGCAGAGCGA TTATCTGGAACTGCTGGTGAAG 27 LAIR1ex +LS1 aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSLY SDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQ SDYLELLVK 28 LAIR1ex +LS1 GAGGACCTGCCAAGACCCAGCATCTCCGCAGAACCTGG nucl GACCGTGATTCCACTGGGCTCCCACGTGACATTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACTTTTAGGCTGGAGCG CGAATCTCGAAGTCTGTACTCCGACACAGAGGACGTGAG CCAGGCCTCACCAAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAACGCCGGCCCTTACAGATG CATCTACTATAAGCCCCCTAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 29 LAIR1ex +LS1R EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSLY aa SDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPRKWSEQ SDYLELLVK 30 LAIR1ex +LS1R GAGGACCTGCCCCGCCCTAGCATCTCCGCAGAACCAGG nucl GACCGTGATTCCCCTGGGCTCCCACGTGACATTCGTCTGC AGGGGCCCCGTGGGAGTCCAGACTTTTAGGCTGGAGCG CGAATCTCGAAGTCTGTACTCCGACACCGAGGACGTGAG CCAGGCCTCACCTAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAACGCCGGCCCTTACAGATG CATCTACTATAAGCCAAGAAAATGGTCAGAGCAGAGCGA TTATCTGGAACTGCTGGTGAAG 31 LAIR1ex +LS1S2R EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSLY aa SDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKSRKWSEQ SDYLELLVK 32 LAIR1ex +LS1S2R GAGGACCTGCCCCGCCCTAGCATCTCCGCAGAACCAGG nucl GACCGTGATTCCCCTGGGCTCCCACGTGACATTCGTCTGC AGGGGCCCCGTGGGAGTCCAGACTTTTAGGCTGGAGCG CGAATCTCGAAGTCTGTACTCCGACACCGAGGACGTGAG CCAGGCCTCACCTAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAACGCCGGCCCATACAGATG CATCTACTATAAGAGCAGAAAATGGTCAGAGCAGAGCGA TTATCTGGAACTGCTGGTGAAG 33 LAIR1ex +LS1T aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSLY SDTEDVSQTSPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQ SDYLELLVK 34 LAIR1ex +LS1T GAGGACCTGCCAAGACCCAGCATCTCCGCCGAACCTGG nucl GACTGTGATTCCACTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTCTGTACTCCGACACCGAGGACGTGAG CCAGACATCACCCAGCGAGTCCGAAGCCCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAACGCTGGCCCTTACAGATG CATCTACTATAAGCCCCCTAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 35 LAIR1ex +LS1TR EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSLY aa SDTEDVSQTSPSESEARFRIDSVSEGNAGPYRCIYYKPRKWSEQ SDYLELLVK 36 LAIR1ex +LS1TR GAGGACCTGCCTAGACCTAGCATCTCCGCCGAACCAGGG nucl ACTGTGATTCCCCTGGGCTCCCACGTGACCTTCGTCTGCA GAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCGC GAATCTCGAAGTCTGTACTCCGACACCGAGGACGTGAGC CAGACATCACCTAGCGAGTCCGAAGCCCGGTTCAGAATC GACTCTGTCAGTGAAGGAAACGCTGGCCCTTACAGATGC ATCTACTATAAGCCAAGAAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 37 LAIR1ex +LS1TS2 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSLY R aa SDTEDVSQTSPSESEARFRIDSVSEGNAGPYRCIYYKSRKWSEQ SDYLELLVK 38 LAIR1ex +LS1TS2 GAGGACCTGCCTAGACCTAGCATCTCCGCCGAACCAGGG R nucl ACTGTGATTCCCCTGGGCTCCCACGTGACCTTCGTCTGCA GAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCGC GAATCTCGAAGTCTGTACTCCGACACCGAGGACGTGAGC CAGACATCACCTAGCGAGTCCGAAGCCCGGTTCAGAATC GACTCTGTCAGTGAAGGAAACGCTGGCCCATACAGATGC ATCTACTATAAGAGCAGAAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 39 LAIR1ex +LS2R EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSLY aa NDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKSRKWSE QSDYLELLVK 40 LAIR1ex +LS2R GAGGACCTGCCCCGCCCTAGCATCTCCGCAGAACCAGG nucl GACCGTGATTCCCCTGGGCTCCCACGTGACATTCGTCTGC AGGGGCCCCGTGGGAGTCCAGACTTTTAGGCTGGAGCG CGAATCTCGAAGTCTGTACAACGACACCGAGGACGTGAG CCAGGCCTCACCTAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAATGCCGGCCCATACAGATG CATCTACTATAAGTCTAGAAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 41 LAIR1ex +LT aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSLY NDTEDVSQTSPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSE QSDYLELLVK 42 LAIR1ex +LT nucl GAGGACCTGCCAAGACCCAGCATCTCCGCCGAACCTGG GACTGTGATTCCACTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTCTGTACAACGACACCGAGGACGTGAG CCAGACATCACCCAGCGAGTCCGAAGCCCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAATGCTGGCCCTTACAGATG CATCTACTATAAGCCCCCTAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 43 LAIR1ex +R aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY NDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPRKWSE QSDYLELLVK 44 LAIR1ex +R nucl GAGGACCTGCCCCGCCCTAGCATCTCCGCAGAACCAGG GACTGTGATTCCCCTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTACCTACAACGACACAGAGGACGTGAG CCAGGCCTCACCTAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAATGCCGGCCCTTACAGATG CATCTACTATAAGCCAAGAAAATGGTCAGAGCAGAGCGA TTATCTGGAACTGCTGGTGAAG 45 LAIR1ex +S1 aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY SDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQ SDYLELLVK 46 LAIR1ex +S1 nucl GAGGACCTGCCAAGACCCAGCATCTCCGCAGAACCTGG GACTGTGATTCCACTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTACCTACTCCGACACAGAGGACGTGAG CCAGGCCTCACCCAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAACGCCGGCCCTTACAGATG CATCTACTATAAGCCCCCTAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 47 LAIR1ex +S1R aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTERLERESRSTY SDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPRKWSEQ SDYLELLVK 48 LAIR1ex +S1R GAGGACCTGCCCCGCCCTAGCATCTCCGCAGAACCAGG nucl GACTGTGATTCCCCTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTACCTACTCCGACACAGAGGACGTGAG CCAGGCCTCACCTAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAACGCCGGCCCTTACAGATG CATCTACTATAAGCCAAGAAAATGGTCAGAGCAGAGCGA TTATCTGGAACTGCTGGTGAAG 49 LAIR1ex +S1S2R EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY aa SDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKSRKWSEQ SDYLELLVK 50 LAIR1ex +S1S2R GAGGACCTGCCCCGCCCTAGCATCTCCGCAGAACCAGG nucl GACTGTGATTCCCCTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTACCTACTCCGACACAGAGGACGTGAG CCAGGCCTCACCTAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAACGCCGGCCCATACAGATG CATCTACTATAAGAGCAGAAAATGGTCAGAGCAGAGCGA TTATCTGGAACTGCTGGTGAAG 51 LAIR1ex +S1T aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY SDTEDVSQTSPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQ SDYLELLVK 52 LAIR1ex +S1T GAGGACCTGCCAAGACCCAGCATCTCCGCCGAACCTGG nucl GACTGTGATTCCACTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTACCTACTCCGACACAGAGGACGTGAG CCAGACCTCACCCAGCGAGTCCGAAGCCCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAACGCTGGCCCTTACAGATG CATCTACTATAAGCCCCCTAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 53 LAIR1ex +S2 aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY NDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKSPKWSE QSDYLELLVK 54 LAIR1ex +S2 nucl GAGGACCTGCCCAGACCTAGCATCTCCGCAGAACCAGG GACTGTGATTCCCCTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTACCTACAACGACACAGAGGACGTGAG CCAGGCCTCACCTAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAATGCCGGCCCTTACAGATG CATCTACTATAAGTCTCCAAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 55 LAIR1ex +S2R aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY NDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKSRKWSE QSDYLELLVK 56 LAIR1ex +S2R GAGGACCTGCCCCGCCCTAGCATCTCCGCAGAACCAGG nucl GACTGTGATTCCCCTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTACCTACAACGACACAGAGGACGTGAG CCAGGCCTCACCTAGCGAGTCCGAAGCTCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAATGCCGGCCCATACAGATG CATCTACTATAAGTCTAGAAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 57 LAIR1ex +T aa EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY NDTEDVSQTSPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSE QSDYLELLVK 58 LAIR1ex +T nucl GAGGACCTGCCAAGACCCAGCATCTCCGCCGAACCTGG GACTGTGATTCCACTGGGCTCCCACGTGACCTTCGTCTGC AGAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCG CGAATCTCGAAGTACCTACAACGACACAGAGGACGTGAG CCAGACCTCACCCAGCGAGTCCGAAGCCCGGTTCAGAAT CGACTCTGTCAGTGAAGGAAATGCTGGCCCTTACAGATG CATCTACTATAAGCCCCCTAAATGGTCAGAGCAGAGCGAT TATCTGGAACTGCTGGTGAAG 59 LAIR1ex +TS2R EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY aa NDTEDVSQTSPSESEARFRIDSVSEGNAGPYRCIYYKSRKWSE QSDYLELLVK 60 LAIR1ex +TS2R GAGGACCTGCCTAGACCTAGCATCTCCGCCGAACCAGGG nucl ACTGTGATTCCCCTGGGCTCCCACGTGACCTTCGTCTGCA GAGGCCCCGTGGGAGTCCAGACCTTCCGGCTGGAGCGC GAATCTCGAAGTACCTACAACGACACAGAGGACGTGAGC CAGACCTCACCTAGCGAGTCCGAAGCCCGGTTCAGAATC GACTCTGTCAGTGAAGGAAATGCTGGCCCATACAGATGC ATCTACTATAAGTCTAGAAAATGGTCAGAGCAGAGCGATT ATCTGGAACTGCTGGTGAAG

[0539] The 20 fusion proteins were expressed in HEK293 cells and the binding to P. falciparum was assessed by staining IEs, as described in Example 1. The results are shown in FIG. 11. These results show that native human LAIR-1 (LAIR1ex) does not bind to infected IEs and that at least one of the mutations T67L (L); N69S (S1); A77T (T); P106S (S2) and P107R (R) is necessary for gaining binding to IE.

Example 10

Influence of the Mutations of LAIR-1 Fragment on Binding to Collagen

[0540] Native human LAIR-1 is well-known to bind collagen, in particular via its extracellular domain (T. Harma C. Brondijk, Talitha de Ruiter, Joost Ballering, Hans Wienk, Robert Jan Lebbink, Hugo van Ingen, Rolf Boelens, Richard W. Farndale, Linde Meyaard, and Eric G. Huizinga (2010): Crystal structure and collagen-binding site of immune inhibitory receptor LAIR-1: unexpected implications for collagen binding by platelet receptor GPVI. Blood 115:7). To identify whether the five mutations influence binding to collagen, the 20 fusion proteins of Example 9 were expressed in HEK293 cells and the binding to collagen was assessed by ELISA. Briefly ELISA plates were coated with Collagen type 1, blocked with PBS 1% BSA, followed by incubation with supernatants and a secondary anti-human (Fc-specific) antibody for detection. The results are shown in FIG. 12. These results show that in particular mutation P107R appears to deteriorate binding to collagen (FIG. 12).

Example 11

Identification of the P. falciparum Antigen(s) Recognized by MGD21

[0541] To identify the antigen(s) recognized by the LAIR1-containing antibodies, stable P. falciparum 3D7 lines, which were enriched (3D7-MGD21) or depleted (3D7-MGD21.sup.) of MGD21 reactivity were generated.

[0542] To investigate MGD21 binding to erythrocyte ghosts and MGD21 immunoprecipitates (IP) prepared from 3D7-MGD21.sup.+ and 3D7-MGD21.sup. IEs, a western blot was performed. Controls included uninfected erythrocytes (uEs) and immunoprecipitates with an irrelevant antibody (BKC3). Anti-human IgG was used as the secondary antibody, resulting in detection of antibodies used for immunoprecipitation alongside antigens of interest. As shown in FIG. 13, western blot analysis revealed two specific MGD21-reactive bands of 40-45 kilodaltons (kDa) in erythrocyte ghosts and in MGD21 immunoprecipitates prepared from 3D7-MGD21 IEs.

[0543] Next, analysis of the MGD21 immunoprecipitates by liquid chromatography coupled with mass spectrometry (LC-MS) was performed. As shown in FIG. 14, this experiment revealed that a member of the A-type RIFIN family (PF3D7_1400600 was significantly enriched in 3D7-MGD21.sup. immunoprecipitates as compared to 3D7-MGD21.sup. immunoprecipitates (log.sub.2 fold change >2; P<0.01). Moreover, RIFIN expression levels in erythrocyte ghosts prepared from 3D7-MGD21.sup.+ and 3D7-MGD21.sup. IEs revealed that PF3D7_1400600 and a second A-type RIFIN (PF3D7_1040300) were also present in 3D7-MGD21.sup.+ but not in 3D7-MGD21.sup. ghosts in the absence of immunoprecipitation (FIG. 15). In contrast, four other RIFINs, including one recently characterized for its capacity to induce rosetting (PF3D7_0100400), were detected in similar amounts in both 3D7-MGD21.sup.+ and 3D7-MGD21.sup. ghosts (FIG. 15).

[0544] In the next step, recognition of 3D7-MGD21.sup.+ IEs and 3D7-MGD21.sup. IEs by other broadly reactive antibodies from donors C (MGC1, MGC2, MGC4, MGCS, MGC17, MGC26, MGC28, MGC29, MGC34) and D (MGD21, MGD39, MGD47, MGD55) were investigated. BKC3 was used as negative control antibody. As shown in FIG. 16, this experiment revealed that enrichment for 3D7-MGD21.sup.+ IEs greatly increased recognition by all the other broadly reactive antibodies from donor D tested and, notably, by two broadly reactive antibodies from donor C. These results suggest that these antibodies recognize the same antigens. Similar results were also obtained with the Kenyan isolate 9605 (FIG. 17A-B).

[0545] The binding of the LAIR1-containing antibodies to specific RIFINs was determined by use of CHO cells transfected with PF3D7_1400600 and PF3D7_1040300, PF3D7_0100400, PF3D7_0100200 and PF3D7_1100500. As shown in FIG. 18A, this experiment confirmed the finding that MGD21 stained CHO cells transfected with the candidate antigens PF3D7_1400600 and PF3D7_1040300, but not with irrelevant RIFINs that were similarly expressed (PF3D7_0100400 and PF3D7_0100200) or not detected (PF3D7_1100500) in 3D7-MGD21.sup.+ and 3D7-MGD21.sup. ghosts. FIG. 18B shows MGD21 and BKC3 staining of CHO cells transfected with a specific (PF3D7_1400600) or an irrelevant (PF3D7_0100200) RIFIN, confirming that the specificity of the binding of MGD21 to the specific RIFIN PF3D7_1400600.

[0546] Furthermore, CHO cells were transfected with a specific (PF3D7_1400600) or an irrelevant (PF3D7_0100200) RIFIN as well as with a RIFIN chimaera containing the constant region of PF3D7_0100200 and the variable region of PF3D7_1400600 and a RIFIN chimaera containing the constant region of PF3D7_1400600 and the variable region of PF3D7_0100200. MGD21 and an Fc fusion protein containing the MGD21 LAIR1 domain stained only those CHO cells, which were transfected with the specific RIFIN PF3D7_1400600 or with the RIFIN chimaera containing the constant region of PF3D7_0100200 and the variable region of PF3D7_1400600, but not cells transfected with the inverse chimaera. Results are shown in FIG. 19, indicating that MGD21 binds to the variable region.

[0547] Collectively, the results obtained in Example 11 indicate that the LAIR1-containing antibodies recognize specific members of the RIFIN family in different P. falciparum isolates.

[0548] In particular, these results identify RIFIN PF3D7_1400600 (amino acid sequence according to SEQ ID NO: 105, nucleotide sequence according to SEQ ID NO: 106) as one major target of the mutated LAIR-1 fragment in P. falciparum and RIFIN PF3D7_1040300 (amino acid sequence according to SEQ ID NO: 538, nucleotide sequence according to SEQ ID NO: 539) as another target of the mutated LAIR-1 fragment in P. falciparum.

[0549] Since RIFINs are highly polymorphic in different strains and the mutated LAIR-1 fragment according to the present invention binds to erythrocytes infected by different P. falciparum strains, it is anticipated that the mutated LAIR-1 fragment according to the present invention will recognize additional RIFINs.