Structural characterization of broadly neutralizing human monoclonal antibodies against the CD4 binding site of HIV-1 gp120

MolecularImmunology, Vol. 31, No. 15, pp. 1149-I 160, 1994 Pergamon

0161-~~~2-1

Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0141-58~194 $7.00 + 0.00

STRUCTURAL CHARACTERIZATION OF BROADLY NEUTRALIZING HUMAN MONOCLONAL ANTIBODIES AGAINST THE CD4 BINDING SITE OF HIV-l gp120 JESSAMYN BAGLEY,* PATRICK J. DILLON,? CRAIG ROSEN,? JAMES ROBINSON,$ JOSEPH SODROSKI* and WAYNE A. MARASCO*§ *Division of Human Retrovirology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115, U.S.A.; THuman Genome Sciences, 9620 Medical Center Drive, Suite 3900, Rockviile, MD 20850, U.S.A.; and department of Pediatrics, University of Connecticut Health Center, 263 Fa~ington Avenue, Fa~ington, CT 06030, U.S.A. (First received 27 December 1993, accepted in revised form 2 June 1994) Abstract-The human monoclonal antibodies (mAbs) 15e and 2 1h are derived from HIV- 1-infected individuals. They block CD4 binding, recognize conformation-dependent discontinuous epitopes on gp120 and neutralize a broad range of laboratory strains and primary isolates of HIV-l. To determine if a structural basis for neutralization could be identified, analysis of these CDCbinding site anti-gp120 human mAbs was performed, common features and differences were identified and a comparison was made with FlO5, a previously reported CD4-binding site anti-gp120 human mAb. The 15e and 21h mAb heavy chains are derived from different V region genes, i.e. V2-1 and VDP35, which are members of the V,IV and V,III families, respectively. Analysis of the genes encoding the heavy chain complementa~ty determining region (CDR) 3 revealed that both mAbs show a long D, segment of similar size that could arise from PD fusions of the dxpl/dlrl and daudi/d22-12 germline DH genes along with use of the Jx6 and JHs germline segments. Similarly, the 1Se and 21h light chains are derived from different V region genes, i.e. Hum01/012 and Humlv318, that are IIIa gene familes, respectively. These V genes are rearranged with members of the VkappaIand Vlambda J kappa1 and JlambdaZ 8ermline genes. For both mAbs, the pattern of replacement mutations in the V region genes of the heavy and light chains is consistent with a process of somatic mutation and antigen-driven clonal selection. By comparing the CDRs of 15e, 21h and Fl05, eight positions in the rearranged heavy chains and two positions in the rearranged light chains were found to have identical amino acids. These studies suggest that there is no absolute restriction in the use of V region germline genes and form the foundation for understanding the humoral immune response to the CDCbinding site of gp120. Key words: AIDS, passive immunization, variable complementarity determining regions (CDRs).

INTRODUCTION Nearly all neutralizing antibodies in the sera of HIV- l-infected humans are directed against envelope glycoproteins gpf20 and gp41 and particularly against the external glycoprotein gp120 (Ho et al., 1987; Steimer

et al., 1991; Moore and Ho, 1993; Kang et al., 1991; Chamat et al., 1992). The binding of serum antibodies reacting with the HIV-l gp120 can neutralize viral infection at two major sites: the V3 loop and the discontinuous epitope that encompasses the CDCbinding region (Kang et al., 1991; Chamat et al., 1992). Synergy between the V3 loop and CD4-binding site antibodies for the neutralization of HIV-l has also been reported (Buchbinder et al., 1992; Tilley et al., 1992). §Author to whom corres~nden~ should be addressed. Abbreviation: CDRs, complementa~ty determining regions.

region gene segments,

B-cell repertoire,

The V3 loop antibodies appear soon after HIV-l infection and are generally more effective than non-V3 loop antibodies in neutralizing a specific HIV-1 isolate (Rusche et al., 1988; Goudsmit et al., 1988; Palker et al., 1988). These antibodies are frequently strain specific (Kang et at., 1991; Gorney et al., 1993) as envelope glycoprotein variation can allow virus to escape neutralization (M&eating et a/., 1989; Looney et al., 1988). Conversely, the CDCbinding site antibodies appear later in infection (Ho et al., 1987; Steimer et al., 1991; Profy et al., 1990) and the CDCbinding region of gp120 is well conserved, although not invariant. Antibodies to this region, both polyclonal antibodies purified from HIV-Iinfected patients sera and human mAbs derived from HIV-l-infected patients, neutralize many strains of HIV-l although not all CD4-binding site antibodies neutralize a diverse range of HIV-l strains equally well (Ho et al., 1987; Steimer et al., 1991; Kang et al., 1991; Tilley et al.,

1149

J. BAGLEY rt al.

1150

1991; Posner et al., 1991, 1993; Ho et al., 1991; Karwowska et al., 1992). The CDCbinding site on gp120 is discontinuous and involves four different regions of the glycoprotein that are brought in close proximity by secondary folding (Olshevsky et al., 1990; Thali et al., 1991, 1992). The CD4-binding site on gp120 does not represent a single epitope but instead appears to represent an “epitope cluster” as defined by several different criteria. First, anti-idiotype antibodies raised against affinity purified CD4-binding site antibodies from pooled HIV-linfected patient sera define different populations of CD4-binding site antibodies with similar CDCblocking activity but exhibit different spectra of neutralizing activities against multiple strains of HIV-l (Chamat et al., 1992). Second, a panel of CD4-binding site recombinant human Fab fragments generated through repertoire cloning of bone marrow cells from an asymptomatic HIV-l -infected patient showed a discordance between their ability to compete with soluble CD4 for binding to gp120 and their ability to neutralize the virus effectively (Burton et al., 1991; Barbas ef al., 1992). Third, studies using a panel of gp120 mutants show that changes in amino acids located within seven discontinuous, conserved regions of gp120 affect antibody binding. The pattern of sensitivity to amino acids changes in these seven regions differed for each antibody studied and also differed from that of the CD4 glycoprotein (Thali et al., 1991, 1992). The mechanism for neutralization of HIV-l by the CD4-binding site anti-gp120 antibodies may involve factors other than CD4 receptor blockage (Barbas et al., 1992). To determine if a structural basis for neutralization could be identified, an analysis of two well characterized, broadly neutralizing, CD4-binding site anti-gp120 human mAbs was performed. Common features and differences were identified and a comparison was made with F105, a previously reported CD4-binding site anti-gp120 human mAb (Marasco et al., 1992). Specifically, germline gene usage was established to determine if there is a restricted use of heavy and light chain V genes in the CD4-binding site anti-gp120 antibody response. Second, the pattern of somatic mutations was documented to establish if these antibodies arose from an antigen-driven selection process. Third, by comparing these broadly neutralizing mAbs an attempt was made to identify a consensus sequence among the heavy and light chain CDRs that allows for efficient HIV-I neutralization.

MATERIALS

AND METHODS

ing the method of Gubler and Hoffman in a 30~1 reaction with 2 pg of total RNA using oligo (dT) priming (Gubler and Hoffman, 1983). The temperatures used for PCR ampli~cation were: melt: 94°C I min; primer anneal: 52°C 2 min; primer extension: 72”C, 2 min. Two 1 min ramps and one 2 min ramp between primer annealing and primer extension were used. PCR fragments were gel purified on ethidium bromide stained 2% agarose gels. The appropriate band was excised, Gene Cleaned (Bio,lOl Inc., La Jolla, CA), restriction enzyme digested and cloned into pSLll80 (Pharmicia LKB, Biotech. Inc., Piscataway, MJ) using SURE bacteria (Stratagene Inc., La Jolla, CA). At least three separate transformants were sequenced using both forward and reverse vector based sequencing primers (Marasco ef al., 1992). The primer used to amplify the 15e heavy and light chains was identical to that published by Larrick et al., 1989. The 5’ primer for the heavy chain was the Group HS3 immunoglobulin leader primer: GGGAA~CATG(A/G)A(A/C)(A~C)(A~T)ACT(G~T) TG(G~T)(A/T)~C~G)C(A/T)(C~T)(C/G)CT~C~T)CTG and the 3’ primer was designed to correspond to residues 120-12.5 of IgG,, CCAAGCTTCTGCCAGGGGGAAGACCGA. The 5’ primer used to amplify the 15e light chain was immunoglobulin leader primer GG GAATTCATGGACA~rG(A~G)(A~G)(A/G)(A~G~T)(C/T)CC(A,‘C,‘T)(A/C/G)G(C/T)(G/’T)CA(C,’G)CTT, and the 3’ primer was CCAAGCTTCATCAGATGGCGGGAAGAT. The 5 and 3’ heavy chain primers contained the cloning sites (underlined) EcoRI and HindIII, respectively. They were cloned into the piasmid Bluescript (Stratagene, La Jolla, CA) and sequenced as described. The primer pairs used to amplify the 21h heavy and light chains were designed with degenerate sites based on the amino acid and codon usage of the human J regions (Kabat et al., 1991). The heavy chain was amplified using a 33 bp 5’ V, primer and a 30 bp 3’ J, primer. The V, primer TTGGCCATGGCCCAGGTGCAGCTGCAGGAGTCG has an NcoI site (underlined) 5’ to the first codon of the V, gene. The J, primer TTAGCGCGCTGAGGTGACCGTGACC(A/G)(G/T)GGT is degenerate at two positions and contains both a BssHII site 3’ to the last codon of the J region (left underlined) and an internal BstEII site (right underlined). The light chain was amplified using a 27 bp 5’ Vlambdaand 27 bp 3’ Clambdaprimer. The Vlambdaprimer TTTTCTAGATC(C/T)T(AjC)TGAACTGACTCAG contains two degenerate sites and an Xbai site 5’ to the first codon of the VW,& region. The Ciambda primer ATTTGCGGC_-.__ -.__ CGCTGGTGCAGCCACAGT contains a Not1 site 3’ to codon 113.

cDNA synthesis and pol,ymerase chain reaction ampll$ication qf the 15e and 21 h immunoglobulin genes The 15e formation (Ho et al., were used experiments.

and 2 1h mAbs are derived from EBV transof the PBMCs of an HIV-l-infected patient 1991; Thali et al., 1992) and both cell lines for cDNA synthesis and PCR amplification First strand cDNA was synthesized follow-

DNA

sequence analysis

DNA sequencing was performed using the Applied BioSystem Automated DNA Sequencer, model 373 using Taq Dye Deoxy Terminator Cycle Sequencing (Applied BioSystems, Foster City, CA).

Structural

analysis

of neutralizing

anti-gp120

antibodies

data

sequencing

1151

RESULTS

were entered into the Eugene Biology sequence analysis package (Molecular Information Resource, Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030). The combined GeneBank/EMBL databases were then searched for homology with the V regions of the heavy and light chains using FASTA, a Pearson/Lippman similarity search program. The heavy chain V region was assumed to end at position 94 using the Kabat numbering system (Kabat et al., 1991). The light chain V region was assumed to end at the start of the J region. The combined D-J region of the heavy chain and the V-J region of the light chain was examined for homology with the published J region sequences (Kabat et al., 1991) using the WordSearch program (Deveraux et al., 1984). Because of the nature of V-D-J joining some of the codons at the 5’ end of the germline J region gene may not be present in the rearranged antibody. It was assumed in the case of the 21h mAb that the entire J region was present (Chothia and Lesk, 1987). Putative germline D region sequences were chosen based on length of homology including both the reverse and reverse complement orientations. Where two genes had the same length of homology the sequence with the fewest gaps and base insertions was selected. DNA

monoclonal

Characterization of the expressed V, genes encoding broadly neutralizing, CD4 binding site directed human anti-HIV-1 gp 120 antibodies The complete variable region sequence of each antibody heavy chain is shown in Fig. 1. Figure 2 shows each of the expressed heavy chain variable region genes compared to their likely, respective, germline equivalents. The 15e heavy chain gene is 93.8% identical to the V2-1 germline gene (Sanz et al., 1989), a member of the V,IV gene family (Fig. 2, panel A). The V, region of mAb 21h is derived from a V,III germline gene and appears most closely related to V,,,, germline gene (94.3%) (Genebank accession no. 212337) (Tomlinson et aE., 1992) (Fig. 2, panel C). The V,,,, germline gene is identical to the V22-2B germline gene (Berman et al., 1988). Table 1 summarizes the number, type and location of somatic mutations in the heavy chain V gene segments. These somatic events should result in mutations distributed randomly throughout the V region. Through random chance, 75% of these mutations should result in an amino acid change-a replacement mutation (Jukes and King, 1979). Thus, the number of expected mutations in any part of the antibody molecule can be calculated from the equation n x Rf x S (Ikematsu et al.,

A V, CAa Q

CTG L

CAG Q

CTG L

TCC S

ATC I

AGC S

gaT D

TAT Y

AGT S

GGG G

CTG L

AAG K

CTG L

CAG Q

GAG E

TCG S

GGC G

CCA P

CGA G

CTG L”

GTG

AAG K

CCT P

TCG S

GAG E

ACC T

CTG L

TCC S

CTC L

ACC T

TGC C

ACT T

GTC V

TCT S

GGT G

GGC G

ttT P

AaT l4

TAC Y

TAC Y

TGG ‘W

GGC G

TGG W

ATC I

CGC R

CAG Q

CCC P

CCA P

GGG G

AAG K

GGa, G

CTt L

GAG E

TGG ‘>!

ATT I

GGc G

AGT S

ATC I

TAT Y

AGC S

gCC A

TAC Y

TAt Y

AAg

K

CCG P

TCC S

CTC L

AAG K

AGT S

CGA R

GTC V

ACC T

ATg I(

TCC S

GTc V

GAC D

ACG T

TCC S

AAG K

AAt CAC H

TTC F

TCC S

AcC T

TCT S

GTG V

ACC T

GCC A

GCA A

GAC D

ACG T

GCT A

GTc V

TAT Y

TAC Y

TGT C

GCG A

AGt S

CAA Q

GTC V

TAT Y

AGG R

CCA P

TAT Y

?uC I

TAC Y

T CC S

GTC V

TGG W

J. GGc G

6

GAC D

CAA Q

GGG G

AgC S

ACG T

GTC V

ACC T

GTC V

TCC S

Tfi S

TCT

GGG

GGA

GaC

TTG

GTC

AAG

CCT

GGA

GGG

TCC

CTG

AEA

CTC

TCC

TGT

GCA

GCC

TCT

GGA

TTC

TAC Y

ATG M

AGC S

TGG W

ATC I

CGt R

CAG Q

GCT A

CCA P

GGG G

AAG K

GGG G

CT2 L

GAG E

TGG !,’

GTc V

TCA S

TAC Y

ATT I

AGT S

AGT S

AGT S

aGT AGT taC ATA aAC TAC SSYINYADSVKGRFTVSRDNAENSLYL

GCA

GAC

TCT

GTG

AAG

GGC

CGA

TTC

ACC

gTC

TCC

AGa

GAC

AAC

GCC

gAG

AAC

TCc

CTG

TAT

CTc

CAA Q

GCG A

AGA R

TCA S

TCT S

CAC H

GCT A

TAC Y

TTC F

CGC R

ACT T

GGG G

z

l .*t+**.***

~*‘*“.*CDR1*********‘******

l t*t.****.~*****~******

mC Y

TAC Y

GGT G

ATG M

N

B V, CAG GTG CAG CTG cTc GAG QVQLLESGGDLVKPGGSLTLSCAASGF .*****.‘CDR~***‘*” ACC T

TTC F

ATG M

AGT S

AAC N

GAC D

AGC S

TAC Y

CTG L

CGA R

GCC A

GAc D

GAC D

ACG T

GCC A

GTG V

TiT Y

TAC Y

TGT C

TGG

J, 5 GGC CAg

GGA

ACC

CTG

GTC

AC

GTC

aCC

TCA

*********_***** aaG TTC GAC CCC =FDP’h’GQGTLVTgVTS

t.**..*t.**.****+**

Fig. 1. Complete nucleotide (B) heavy chains. Asterisks

and deduced amino acid sequences of the rearranged 15e (A) and 21 h indicate CDRs. Bold nucleotides (lower case) and bold amino acids (upper case) represent somatic or replacement mutations, respectively, compared to their putative germline genes. Where the primer annealing sequence is difference from the germline sequence, changes are denoted by lower case letters. The individual V, D and J gene segments are separately marked or underlined. Every tenth codon, using the Kabat numbering system (Kabat et al., 1991), is marked with a solid circle.

V

J. BAGLEY et al.

1152 A.

lie CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGC

‘“‘2 - 1

:

15e

:

v2-1 15e

: :

GACACGGCTGTGTATTACTGTGCGAGA -------~~~-C--------------T

DP-35

:

21h

:

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCAGT ~~~~---A---------_______.__----C__--------_.._.~~--------------._~ ‘+“**“CDR1”*

DP-35 2lh

: :

GACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGC -----------------------T_----------_____._.~--..._....C_..._._~_~~-------A-----TA~___A.._.. I***********+*****

DP-35

:

21h

:

GCAGACTCTGTGRAGGGCCGATTCACCATCTCCAGGGACAAGAGCCGAGGAC ~~~~~~~~-~~~--~-----~~-~~~~G~~~~~~~A-_-------G_------C----_C---_--------~-~C~~~..__c...

DP-35 21h

: :

B.

21h

l****‘****“**,*‘*CD~2*,*,***.***

ACGGCCGTGTATTACTGTGCGAGA

Fig. 2. Nucleotide sequence of each expressed heavy chain variable region compared to the nucleotide sequence of the closest germline genes. CDRl and CDR2 are indicated by the asterisks. (A) Nucleotide sequence of the 15e V, compared to the V2-1 (V,IV) germline gene (Lee et al., 1987). (B) Nucleotide sequence of the 21h V, compared to the V,,,, (V22-2B) (V,III) germline gene (Berman et al., 1988; Tomlinson et al., 1992). Dots indicate primer annealing sequence.

1994) where n equals the total number of mutations, Rf equals the expected proportion of replacement mutations (0.75) and S equals the relative size of the region in question (CDR = 0.23, FR = 0.77). When the number of replacement mutations expected and the number of those which are actually present in the antibody differ significantly, it must be due to selection pressure-antigen-driven clonal selection. In the 15e V gene segment there are mutations in 16 codons, 12 of which are expected to result in mutations. In fact, only eight do result in amino acid replacements. The likelihood that this would occur by random chance was calculated by the chi squared test to be p = 0.019.

Consistent with antigen-driven clonal selection, the FR showed a lower number of replacement mutations than expected (3 vs 9) while the CDR had more than expected (5 vs 2). The R/S ratio in the CDRs is 5 whereas in the FRs the R/S ratio is 0.4. For the 21h V gene segment there are mutations in 14 codons, eight of which result in amino acid replacements (p = 0.076). As with 15e, there were fewer replacement mutations in the FR than expected (5 vs 8) and more in the CDR (3 vs 2). All of the mutations in the CDRs result in amino acid replacements whereas in the FRs the R/S ratio is 0.8. In both the heavy chains a greater than expected proportion of replacement mutations are seen in the CDRs

Table I. Summary of somatic changes in the 15e and 21h heavy chain variable regions A. 15e Regions

Mut

R

R/S

VH

16

8

6

5

1 5

CDR

FR

10

3

0.4

B. 21h Regions

Mut

R

R/S

VH CDR

14 3

8 3

1.3 &

FR

11

5

0.8

Position 31 32 33 57 60

Conserved

S>D S>F

P> P>NP

T>A N>K

P>NP P>+

R>S

+ >P

S>N

69 82A 94

I>M S>T

Position

Conserved

54 56 58

T>Y

10 19 69 75 85

Nonconserved

Nonconserved G>S

NP>P

A>N

NP>P

G>D R>T

NP> + >P

K>E

+>-

I>V E>D

Structural analysis of neutralizing A. 158 dlrl 15e

anti-gp120 monoclonal

1153

antibodies

CATAcaccattagtacaatatcct gqTATAQ CaagtcTATAQqcCATAt CaAQTCtAt&ggccagt gttataataaCcAQTCaAa&taWggatc

15e dxpl B. 21h 21h dlr4

t=ctCAcQCT TACTtCcgscgggg gg=agCA QCTqqTACTaCtamazatcct :r ATaTCACQaTTtt~gga~tg~ttap tcATcTCACQcTTacxtcc&zaBggg

daudi 21h 21h d22-12

tcatctc&gcxt&cEccQcACTQQg gtAttaxg&tEttQqACTQQttattatacc

Fig. 3. Nucleotide sequences of 15e and 2 1h D region genes compared to known human Da germline genes. Underlined and bold nucleotides show identity between the antibody D regions and the known D, germline genes.

and a smaller proportion of replacement mutations than expected are seen in the FR.

Analysis of the D, and JH gene segments encoding CDR 3 15e D, segment may represent a D-D fusion product with the dlrl and dxpl germline D, genes (Fig. 3). The 21h D, segment appears to be derived either from the dlr4 germline D, segment alone or may represent a D-D fusion product with the daudi and d22-12 germline D, genes. An analysis of the J, gene segments of the two mAbs reveals that 15e uses the J,, germline gene whereas 21h utilizes the J,, germline gene. Table 2 summarizes the nucleotide and amino acid changes for the J, gene segments. The 15e J, region differs by four nucleotides from that of JH6.The two differences that occur in the CDR of 15e J, result in amino acid changes (1.35 expected) whereas only one of the two differences that occurs in the FR results in an amino acid change (1.65 expected). The 21h J, region differs by seven nucleotides from that of J,,. All three of the differences in the CDR3 result in amino acid changes (one expected) whereas the only nucleotide difference in the FR is silent (two expected). In both the J regions more replacement mutations than expected are found in the CDRs while fewer replacement mutations than expected are found in the FRs. The

Figure 4 compares the heavy chain CDRs of 15e, 21 h and F105 (Marasco, 1992). In position 31, both 15e (as the result of a replacement mutation) and 21h have

aspartic acid. There are four positions of homology at the carboxyl end of the CDR. A tyrosine is found at a similar location in 15e (position 34) and 21h (position 32). In position 33 (position 35 for 1se), tyrosine is found in all three heavy chains. In position 34 for F105 and position 35A for 15e, tryptophan is found and in position 35, serine is found in F105 and 21h. In CDR2, there are 12 positions of identity between F105 and 15e that result from their related V,IV germline genes. Positions 51, 54, 59, 62 and 64 have identical amino acids for all three heavy chains. Only at position 54 for 21h is this identity due to a replacement mutation. Otherwise, the identity is due to germline configuration. There are seven positions where identical amino acids are found between F105 and 21h. Two of these positions, 54 and 58, have replacement mutations in 21h that result in amino acids changes that are identical to the germline amino acids expressed in F105, serine and asparagine, respectively. The five other positions of identity are due to germline configuration. There are five positions of identity between 15e and 21h. In position 54 for 21h this is a result of a replacement mutation.

Table 2. Summary of somatic changes in the 15e and 21h heavy chain J regions A. 15e Region

Mut

JH CDR

R

R/S

4

3

3

2

2

%

Position

Conserved

1OOA 1ooc

Y>S

Y>F

2

1

1

107

T>S

B. 21h Regions

Mut

R

R/S

Position

Conserved

Jti

4

3

3

CDR

3

3

%

FR

1OOD 1OOE 102

FR

1

0

0

Nonconserved P>NP

Nonconserved

N>V W>K S>P

P>NP NP>+ P>NP

1154

J. BAGLEY et al.

CDR II 50

CDR III 95

51

96

52

97

52A 53

98

99

54

55

56

57

58

59

60

61

62

63

64

65

100 100A 100B 1OOC 1OOD 1OOE 1OOF 1OOG 101 102

Fig. 4. Heavy chain CDR comparison among the F 105, 15e and 21h rearranged heavy chains. Bold amino acids represent replacement mutations compared to their respective germline genes. Boxes enclose amino acids in any position where the amino acids were shared between at least two different antibodies.

Thin vertical bars in CDR3 represent D-J boundaries. Numbering numbering system (Kabat et al., 1991).

In CDR3, F105 and 15e are 15 amino acids long whereas 21 h is 14 amino acids long. When maximally aligned, positions 1OOA and 101 have identical amino acids for all three heavy chains, position 100 of 15e as a result of a replacement mutation. Five other positions have identical amino acids for two of the heavy chains. Most notably, position 102 for F105 and 21h has proline as a result of a replacement mutation in the J,, gene segments. In position 1OOE both F105 (as the result of a replacement mutation) and 15e have a tyrosine. Characterization of the expressed broadly neutralizing, CDCbinding anti-HIV-l gp 120 antibodies

VL genes encoding site directed human

The complete variable region sequence of each antibody light chain is shown in Fig. 5. Figure 6 shows each of the expressed light chain variable region genes compared to their putative germline equivalents. The 15e light chain shares 93.7% homology with the Hum02/012 germline gene, a member of the kappa I subgroup of kappa chain genes (Pargent et al., 1991). The 21h light chain has 94.7% homology with the Humlv318 germline gene (Daley et al., 1992), a member of the lambda IIIa subgroup of lambda genes. Table 3 summarizes the number, type and location of somatic mutations in the light chain V gene segments. The 15e gene segment has mutations in 16 codons. Eleven of these mutations result in amino acid replacements (12 expected). The R/S ratio in the CDR is 1.5 whereas in the FR the R/S ratio is 2.6.

is by using the Kabat

In the FR there are fewer, and in the CDR there are more replacement mutations than expected. For the 21h V gene segment there are mutations in 11 codons, eight of which result in amino acid replacements. Both of the mutations in the CDRs result in amino acid replacements whereas in the FRs the R/S ratio is 2. As in the 15e light chain there are fewer and more replacement mutations in the FR and CDR, respectively. Analysis

of the light chain J segments

There are five human J kappagermline genes and five human J ,ambdagermline genes. 15e uses the Jkappa, and 2 1h germline genes. The light chain CDR3 is uses the J ,ambdaZ derived mainly from the V gene segment with the addition of two amino acids at the carboxy end of the CDR that are derived from the joining residues at 5’ end of the light chain J segment. As can be seen in Fig. 5 and Table 4, the most 5’ joining residue of the 15e Jkappagenes has a replacement mutation whereas 21h has a silent mutation in this same position and the second codon of 15e has the 21h J ,ambdagene has a replacement mutation. three FR mutations all of which result in amino acid replacements whereas 21 h has none. Comparative

analysis of the light chain CDRs

Figure 7 compares the light chain CDRs of the three mAbs. A comparison of light chain CDRl shows seven positions of identity between F105 and 15e and all of these positions are in germline configuration. The only

Structural analysis of neutralizing

anti-gp120 monoclonal

1155

antibodies

A l***fCDRl**.*** V” GAa cTg CAG CTG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GtC AGA GTC AaC cTC ACT 'KC CGG GCA AGT CAG I L Q L T Q S P S S L S A S" G V R V N L T C R A S Q l ,*******CDR2*‘****

l*t*****t~t+**++*.tt*~****

AGC ATT gaC AtC TAT 'PTAAAT TGG TAT CAG CAG AAg CCA GGG AAc GCC CCT AAG CTC CT".? ATC TAT GCT GCA TCC AGT TTa S I D I Y L N WY Q Q K P G N A P K L L I Y A A S S L CAg AGT GGG GTC CCA TCA AGG TTC AGT GGC AGT GGg TCT GGG ACA GAc 7°K ACT CTC ACC ATC AGC AGT CX Q S G" P S R F S G S G S G T D F T L T I S S L

CAA C$,TGAA Q R E

**.~,*.**CDR3~*~*'*.*~~~*******,.* J, 1 GAT TIT GCA ACT TAC TAC TGT CAA CAG AGT TcC AGT ACC CCT caG ACG TTC GGC CcA GGG ACC gtG GTG Gt DFATYYCQQSSSTPQTFGPGTVVV'IKR

ATC AAA Cfi

B '*.*CDRl****+'*+**+ V TCt TAT Gas CTG ACT CAG CCA CCC TCG GTG TCA GTG GCC CCA GGA cAG ACG GCC AGG ATT tCC TGT GGG GGA AAC AAC ATT SYELTQPPSVSVAPGQTARISCGGNNI

*t+~t~+t**tt*t****tt***

*,******C~R~~*‘*~*+*‘-*

GGA AGT AtA AGT GTG CAC 'KG TAC CAa CAG AIlG&A G S I S V H Y Y Q Q R P

GGC CAG GCC CCT GTG Cn: GTC GTC TAT GAT GAT AGC GAC CGG CCC G Q A P V L V V Y D D S D R P

TCA GGG ATC CCT GiG CGA TPC TCT GGC TCC AAg TCT GGG AAC AbG GCC AtC CTG ACC ATC AGt cGG GTC GAA G& S G I P E R F S G S K S G N T A I L T I S R V E A

GGG GAT G D

l J 2 CAG GTG TGG GAT AGT ACT AGT GAT CAT TTC GTa aTA TTC GGC CGA GGG ACC AAG CTG ACC GTC W D S T S D H F V I F G G G T X L T V Q V *‘*‘,**CDR3******“**~********~~,,.**~**~**”**

GAG GtC GAC TAT TAC XT E V D Y Y C

m L

Fig. 5. Complete nucleotide and deduced amino acid sequences of the rearranged 15e (A) and 21h (B) light chains. Asterisks indicate CDRs. Bold nucleotides and amino acids represent somatic or replacement mutations, respectively, compared to their putative germline genes. Where primer annealing sequence differs from gerrnline, sequence changes are denoted by lower case letters. The individual

V and J gene segments are separately marked or underlined. Every tenth codon, the Kabat numbering system (Kabat et al., 1991) is marked with a solid circle.

similarity that 21h shares with F105 is at position 30 (position 29 for FlOS) and only at position 31 for 21 h and 15e has a replacement mutation in both chains resulted in an identical amino acid. In CDR2, only position 52 is identical for all three light chains. Three positions have identical amino acids between F105 and 15e (positions 51-53), two positions have identical amino acids between F105 and 21h (positions 52 and 54) and two positions have identical amino acids between 15e and 21h (positions 52 and 56).

using

In CDR3, only position 89 is identical for all three light chains. All three identical amino acids seen between FlO5 and 15e (positions 89, 90 and 97) are in germline configuration. Two identical amino acids are found between F105 and 21h (positions 89 and 92) and at position 92 is the result of a replacement mutation for F105. Three identical amino acids are found between 15e and 21h (positions 89, 93 and 94) and only in position 94 for 21h is this the result of a replacement mutation.

A. 15e ****CJJR1’***++

Vk012/02: GACATCCAGAn;ACCCAGTCTCCATCCTCCCTGTCmCATAG 15ek : __AC_G___C__---------__.__________________.__.___T___.____A.C_____----------------**tat************* *********CDR2**+"' Vk012/02: CATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGG~GCCCCT~GCTCCTGATCTATGC~CATCCAG~TGC~ ____GA__T__----------____________G-------_C_.__.__________-_------------------A__G_ 15ek : * Vk012/02: GTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACT 15ek : _____----------____________-_--G----------_C_______-------------------__G.__.___.__ ++'***,**CDR3~******** Vk012/02: GCAACTTACTACTGTCAACAGAGTTACAGTACCCCT 15ek : ---------_________-------C---_-_____ l

l

8.

21h

V1318 21h

l*"+'*CDRl"**+* : TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCCAGG~GACGGCCAGGATTACC~~GGGG~C~CATTGG ____________________._____c-____._____~___.T__.___________-------: . ................. ++*++++***.t**** +t**ttt*~r*~~D,**+*t _"I\,. : AAGTAAAAGTGTGCACn;GTACCAGCAGAAGCCAGGCCAGGCCAGGCCCCTGTGC~GTCGTCTATGA~ATAGCGACCGGCCCTCAG : _____T___---___________-A-___G______________-----------______------------_-__.__.__ l

V1318 21h VI318 21h V1318 21h

: GGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGCCGGGGATGAGGCC : -------_______-----___.____G-_------------____T------------TC__-----------------__T. l'+*~*"*"******+CDR3****~*** : GACTATTACTGTCAGGTGTGATAGTAGTAGTGATCATC : ___-_----_______________.__c----________---

Fig. 6. Nucleotide sequence of each expressed light chain variable region compared to the nucleotide sequence of the closest germline genes. CDRl, CDR2 and CDR3 are indicated by the asterisks. (A) Nucleotide sequence of the 15e Vkappacompared to the HumO2/012 (VkappaI) germline gene (Pargent et al., 1991). (B) Nucleotide sequence of the 2lh VlsmM2,compared to the Humlv318 (V,,,,,IIIa) germline gene (Daley et al., i992). Dots indicate primer annealing sequence.

1156

J. BAGLEY Table 3. Summary

of somatic

changes

et al.

in the 15e and 21h heavy chain variable

regions

A. 15e Regions

Mut

R

R/S

VK

16 5

11 3

2.2 1.5

CDR

FR

11

8

2.6

Position

30 31 92

VA CDR FR

Mm

R

11 2

8 2

9

6

R/S 2.7 >>>> 2

1

Table 4. Summary A. 15e Regions

of somatic

31 94

S>T

17 22 39 66 72 84

Mut

R

R/S

CDR

4 1

4 1

% %

FR

3

3

%

JK

B. 21h Regions

J, CDR

Mut

R

R/S

2 2

1 1

1 1

changes

A>E

NP>

D>V

->NP

K>N P>R

+ >P NP> +

-

Nonconserved

K>I

+ >NP

K>Q

+ >P

N>K T>I

p>+ P>NP

T>S K>R

A>V

F105 than the previously reported V71-4 germline gene (95.8%) (Marasco et al., 1992). Restricted heavy and light chain V gene use in humans occurs in several autoimmune (Silberstein et al., 1991; Stevenson et al., 1986) and infectious diseases (Davidson et al., 1989; Adderson et al., 1991; Schrieber et al., 1990). This restriction is relative and not absolute so that infrequent use of some V genes still occurs (Davidson et al., 1989; Manheimer-Lory et al., 1991; Paul et al., 1992). The present and our previous study show that 21 h, 15e and F105 mAb heavy chains and light chains are derived from different V region germline genes (Marasco et al., 1992). The 15e and 21h mAbs are derived from the same HIV- 1-infected individual, the 15e mAb having been isolated earlier than the 21h mAb

in the 15e and 21h light chain regions

Position

Conserved

100 103 105

Position

Nonconserved

W>Q Q>P

96

99

P>P>-

T>N I>L

Conserved

This study reports the primary nucleotide and deduced amino acid sequence analysis of two human mAbs that are broadly neutralizing for many laboratory strains and primary isolates of HIV-I and are directed towards the conformationally sensitive CD4-binding site of gp120. The results show that both mAb heavy chains and light chains are derived from different V region germline genes together with individual point mutations in these genes. The F105 heavy chain gene, another CDCbinding site gpl20 antibody, is also a member of the VJV gene family and is most homologous to the V”4.11 germline gene (96.2%) (Sanz et al., 1989). This germline gene has one more nucleotide homology with

S>D S>I

I>L M>L

Position

DISCUSSION

Nonconserved

Y>S

2 4 17 20 21 42 80 B. 21h Regions

Conserved

K>V E>V

Conserved V>I

NP>P P>NP + >NP ->NP

Nonconserved

Structural analysis of neutralizing CDR

antibodies

1157

I

24

25

26

27

27A

28

29

30

31

32

33

34

CDR II 50

CDR

anti-gp120 monoclonal

51

52

53

54

55

56

TIT

89

90

91

92

93

94

95

95A

958

95C 96

97

Fig. 7. Light chain CDR comparison among the 15e and 21h rearranged light chains. Bold amino acids represent replacement mutations compared to their respective germline genes. Boxes enclose amino acids in any position where the amino acids were shared between at least two different antibodies. Numbering is by using the Kabat numbering system (Kabat et al., 1991).

(Thali et al., 1992). The V, family use of three additional

CD4-binding site anti-gpl20 mAbs i.e. GP13, GP44 and GP68 V,V, V,I and V,I, respectively, has been reported (Schutten et ai., 1993). The heavy and light chains of the recombinant CD4-binding site anti-gp120 Fab fragments derived by phage selection also appear to use different families of V region genes primarily VJ, VuIII, I (Barbas et al., 1992, 1993). These Vkappa and V,,,III, data appear to indicate that if restricted heavy and light chain V gene use does occur, it is not absolute. The relationship between these molecular data and the observed cross-reactive idiotypes of CD4-binding site antigp120 serum antibodies remains to be determined (Chamat et al., 1992; Wang et al., 1992). Both the 15e and 21h heavy chain germline genes are used in fetal and adult populations against exogenous and autoantibodies. The 15e heavy chain germline gene, for example, has been found in an IgM antibody recovered from a patient with X-linked agammaglobulinemia (Mortari et al., 1991) as well as a high affinity monoreactive IgM rheumatoid factor (Harindranath et al., 1991). The 21h heavy chain also shares a high degree of homology with two rearranged heavy chains recovered from cord blood cells (Mortari et al., 1992). Anti-DNA (Manheimer-Lory et ai., 1991) and anti-rabies virus monoclonal antibodies (Ikematsu et al., 1993) have also been shown to utilize the 22-2B heavy chain germline gene. In both the 15e and 21h heavy chains, the FR have lower numbers of replacement mutations than would be expected from random distribution. This suggests that mutations are selected against in favor of maintaining the protein structure. The CDRs on the other hand show evidence of selection pressure for mutations by tending to have a greater number of replacements than would be expected by chance. Both the 15e and 21h light chains

show a similar tendency towards in the CDR and silent mutations

replacement mutations in the FR though not

to as great a degree as the heavy chains. Taken together, these data strongly suggest an antigen driven process of clonal selection. In addition, changes in the Vu segment must be considered in the context of the overall selection forces that act on the heavy chains. The power of antigen selection can make D-D fusions, which occur at a lower frequency than normal joining, biolo~~ally significant. One notable similarity among the 15e, 21h and F105 heavy chains is the size of the CDR3 region (Marasco et al., 1992). The heavy chain CDR3 is known to be important in determining antigen specificity (Kabat et al., 1991; Lane et al., 1983; Kabat and Wu, 1991). Phenylalanine 43 has been implicated in binding in several mutagenesis studies (Arthos et al., 1989; Brodsky et al., 1990) and crystallography studies of human CD4 (Ryu et al., 1990; Wang et al., 1990), and involvement of the phenyl group is also suggested by peptide inhibitor studies (Ryu et al., 1990; Wang et al., 1990). Phenylalanine is also a common element in the heavy chain CDR3 region of 15e, 21h and F105 mAbs. The significance of this relationship remains to be investigated. When the 15e, 21h and FlO5 mAbs are compared to thirty-six CD4 binding site antibodies obtained by repertoire cloning from an asymptomatic individual (Barbas et al., 1993) very little homology can be seen. Fourteen of the heavy chains are V,I and fourteen are V,III with less than 90% homology with the nearest human germline Vu111 gene. The only exception to these are eight V,III which have 93% homology with the 2Pl germline gene. This germline gene has less than 83% homology with the V,III 21h mAb. When the VJII heavy chain CDRs are compared to those of 21h, the maximum homology is 25%, all due to germline residues (Yamada et al., 1991). All of these recombinant Fab fragments are derived from bone marrow cells and their ability to neutralize is not known. The Fab 13 light chain in group 2, on the other hand, was suggested to have unique features in its CDRs that could be responsible for neutralization activity. A comparison with the light chain of Fab 13 showed a common arg31 in CDRl and asn93 in CDR3 with F10.5, a common asp30 and ile31 in CDRl with 15e and a common ile31 in CDRl with 21h (Barbas et al., 1992). In addition, the germline gene closest to the F105 V,, Humvk325, is the same used in 13/33 of the light chains. As has been pointed out however (Barbas et al., 1993), this may not be unique to HIV-seropositive indi~duals. In conclusion, the pattern of replacement mutations in these commonly used V region genes indicates that an antigen driven clonal selection process underlies the emergence of HIV-l gp120 CD4-binding site specific monoclonal antibodies capable of neutralizing a broad range of HIV-I strains. Acknowledgements-We thank the Core Facility at the DanaFarber Cancer Institute for performing the DNA sequencing. A portion of this work was funded by American Foundation

1158 for AIDS Research (001252-9-RG) and the National of Health, K08CAOl507 and AI31783.

J. BAGLEY Institutes

et ul.

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