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Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.

Protein S is a cofactor of activated protein C; together they function as a regulator of blood coagulation. A human liver cDNA library constructed in bacteriophage Xgtll was screened with DNA fragments from a full-length bovine cDNA clone encoding protein S. Several cDNA clones were isolated and sequenced. The combined cDNA sequences encoded the mature protein and 15 residues ofthe leader sequence when compared to bovine protein S. Human protein S is a single-chain protein consisting of 635 amino acids with 82% homology to bovine protein S. After an NH2-terminal y carboxyglutamic acid-containing region, there is a short region with thrombin-sensitive bond(s), followed by a region with four repeat sequences that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the COOH-terminal portion of human protein S does not show any resemblance to serine proteases.Protein S is a single-chain plasma glycoprotein that undergoes vitamin K-dependent y-carboxylation during its biosynthesis (1-4). The concentration of protein S in human blood plasma is -25 mg/liter (4). Both human and bovine protein S have been purified from plasma, and recently the amino acid sequence of the bovine protein was established (5). Unlike the vitamin K-dependent clotting factors, protein S is not a proenzyme to serine protease but functions as a cofactor to activated protein C (6, 7). Patients with hereditary protein S deficiency, as well as those with protein C deficiency, suffer from a predisposition to venous thrombosis (8, 9). In addition to its established role as a cofactor to activated protein C, a regulatory role for protein S in the complement system has been suggested based on the observation that half of protein S in plasma is in a 1:1 complex with complement component C4b-binding protein (10,11).Bovine protein S has 11 y-carboxyglutamic acid (Gla in sequences) residues (12). In addition, acid hydrolysates ofthe bovine protein have been found to contain 03-hydroxyaspartic acid (Hya in sequences) (13-15), which is located in regions homologous to the epidermal growth factor (EGF) precursor (16) in all vitamin K-dependent plasma proteins except prothrombin (13-15, 17, 18); its function is unknown. We now report the isolation and sequence of human cDNA clones that code for mature human protein S. MATERIALS AND METHODSA human fetal liver cDNA library in phage Xgtll was prepared by a modification of the procedure of Gubler and Hoffman (19) similar to that described by Lapeyre and Amalric (20). The library contained >6 x 107 recombinants with inserts averaging 1800 nucleotides in length. Approximately 2 x 107 plaques from the amplified library were screened by standard techniques (21,22). RNA blot-hybridization analysis was conducted by standard methods (23). Nick-translated DNA fragments from a bovine protein S cDNA clone, pBLS-2400 (5), and a human protein C cDNA clone (N. Capalucci and R.W., unpublished data) were used as probes.For sequence analysis, 200...

We have mapped a signal sequence for mRNA 3'-end formation in Saccharomyces cerevisiae by using a Drosophila melanogaster DNA segment that complements a yeast adenine-8 mutation. That the 3' end of the transcript in S. cerevisiae nearly coincides with that in D. melanogaster is consistent with the possibility that mRNA termini are similarly determined in both organisms. Deletion analysis reveals that the complete signal is no more than 21 base pairs long. Part of the signal is the sequence TTTTTATA, which is seen in the termination region of several yeast genes. TTTTTATA appears to be able to act autonomously as a partial termination signal. The efficiency of the complete signal is affected by substitution of sequences downstream from it. This modulation of the effect of a signal is consistent with termination in S. cerevisiae, resembling rhodependent termination in bacteria.Until recently, transcription termination in eucaryotes has been poorly understood, in part because of the rapid processing that occurs at the 3' ends of transcripts for proteincoding genes. Processing usually involves cleavage of a precursor followed by polyadenylation in higher eucaryotes (6, 21). Histone mRNA 3' ends which lack polyadenylic acid were once thought to result from transcription termination (3) but now appear to be generated by processing (2, 7). The biochemical elucidation of mRNA 3' processing should be possible with the recent development of a cell-free system (23). In contrast to processing, actual transcription termination, which can occur at a considerable distance downstream from the mRNA 3' end (6, 27), has been relatively refractory to analysis.In Saccharomyces cereisisiae, polyadenylic acid addition and transcription termination appear to be coupled, as essentially all transcripts from protein-coding genes are polyadenylated [poly(A)+] (29). Therefore, the strong possibility exists that the study of 3'-end formation of an mRNA in yeast cells will shed light on the actual transcription termination event. Termination is known to be important in the regulation of a variety of bacterial operons (reviewed in references 17 and 24) and appears to be involved in the regulation of at least one eucaryotic gene (9, 10, 28). We have been investigating the sequence determinants of transcription termination on a DNA segment in S. cerev,isiae. This segment is derived from Drosophila melanogaster and complements a yeast adenine-8 mutation (15). We showed previously that mRNA 3' ends actually occur 50 to 90 base pairs (bp) downstream from a control signal, the 3' boundary of which we mapped precisely (13). Here we show that this signal is no more than 21 bp long. We also present evidence suggesting that an 8-bp portion of this signal promotes partial termination. It

We have investigated transcription termination on a segment of Drosophila DNA that complements a yeast adenine-8 mutation. Poly(A)+ RNA transcribed from this segment in yeast terminates at multiple sites clustered just beyond an AAUAAA sequence implicated in polyadenylation of higher eucaryotic messages. Deletion analysis indicates that, in yeast, this sequence is not required for polyadenylation. Rather, transcription termination is signalled by a region that is upstream of the AAUAAA sequence. At least part of the control region appears to be an 8-base pair (bp) sequence also found in the termination control region of the yeast CYC1 gene. Termination sites for the various deletions show a clear sequence preference. These sites occur in clusters at least 50 bp downstream of the control region, suggesting similarities between termination in yeast and p-dependent termination in bacteria.